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1
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Ferrero L, Zhang W, Benhamed M, Crespi M, Ariel F. Non-B DNA in plant genomes: prediction, mapping, and emerging roles. TRENDS IN PLANT SCIENCE 2024; 29:1224-1244. [PMID: 39079769 DOI: 10.1016/j.tplants.2024.06.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Revised: 06/13/2024] [Accepted: 06/26/2024] [Indexed: 11/09/2024]
Abstract
Regulating gene expression in plant development and environmental responses is vital for mitigating the effects of climate change on crop growth and productivity. The eukaryotic genome largely shows the canonical B-DNA structure that is organized into nucleosomes with histone modifications shaping the epigenome. Nuclear proteins and RNA interactions influence chromatin conformations and dynamically modulate gene activity. Non-B DNA conformations and their transitions introduce novel aspects to gene expression modulation, particularly in response to environmental shifts. We explore the current understanding of non-B DNA structures in plant genomes, their interplay with epigenomics and gene expression, and advances in methods for their mapping and characterization. The exploration of so far uncharacterized non-B DNA structures remains an intriguing area in plant chromatin research and offers insights into their potential role in gene regulation.
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Affiliation(s)
- Lucía Ferrero
- APOLO Biotech, Santa Fe de la Vera Cruz, Santa Fe, Argentina
| | - Wenli Zhang
- State Key Laboratory of Crop Genetics and Germplasm Enhancement and Utilization, CIC-MCP, Nanjing Agricultural University, No.1 Weigang, Nanjing 210095, China
| | - Moussa Benhamed
- Université Paris-Saclay, CNRS, INRAE, Université d'Évry, Institute of Plant Sciences Paris-Saclay (IPS2), Bâtiment 630, 91192 Gif-sur-Yvette, France
| | - Martin Crespi
- Université Paris-Saclay, CNRS, INRAE, Université d'Évry, Institute of Plant Sciences Paris-Saclay (IPS2), Bâtiment 630, 91192 Gif-sur-Yvette, France
| | - Federico Ariel
- APOLO Biotech, Santa Fe de la Vera Cruz, Santa Fe, Argentina; Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), CONICET-Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina.
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2
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Rajan-Babu IS, Dolzhenko E, Eberle MA, Friedman JM. Sequence composition changes in short tandem repeats: heterogeneity, detection, mechanisms and clinical implications. Nat Rev Genet 2024; 25:476-499. [PMID: 38467784 DOI: 10.1038/s41576-024-00696-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/19/2024] [Indexed: 03/13/2024]
Abstract
Short tandem repeats (STRs) are a class of repetitive elements, composed of tandem arrays of 1-6 base pair sequence motifs, that comprise a substantial fraction of the human genome. STR expansions can cause a wide range of neurological and neuromuscular conditions, known as repeat expansion disorders, whose age of onset, severity, penetrance and/or clinical phenotype are influenced by the length of the repeats and their sequence composition. The presence of non-canonical motifs, depending on the type, frequency and position within the repeat tract, can alter clinical outcomes by modifying somatic and intergenerational repeat stability, gene expression and mutant transcript-mediated and/or protein-mediated toxicities. Here, we review the diverse structural conformations of repeat expansions, technological advances for the characterization of changes in sequence composition, their clinical correlations and the impact on disease mechanisms.
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Affiliation(s)
- Indhu-Shree Rajan-Babu
- Department of Medical Genetics, The University of British Columbia, and Children's & Women's Hospital, Vancouver, British Columbia, Canada.
| | | | | | - Jan M Friedman
- Department of Medical Genetics, The University of British Columbia, and Children's & Women's Hospital, Vancouver, British Columbia, Canada
- BC Children's Hospital Research Institute, Vancouver, British Columbia, Canada
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3
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Li J, Wang H, Yang W. Tandem MutSβ binding to long extruded DNA trinucleotide repeats underpins pathogenic expansions. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.12.571350. [PMID: 38168405 PMCID: PMC10760016 DOI: 10.1101/2023.12.12.571350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
Expansion of trinucleotide repeats causes Huntington's disease, Fragile X syndrome and over twenty other monogenic disorders1. How mismatch repair protein MutSβ and large repeats of CNG (N=A, T, C or G) cooperate to drive the expansion is poorly understood. Contrary to expectations, we find that MutSβ prefers to bind the stem of an extruded (CNG) hairpin rather than the hairpin end or hairpin-duplex junction. Structural analyses reveal that in the presence of MutSβ, CNG repeats with N:N mismatches adopt a B form-like pseudo-duplex, with one or two CNG repeats slipped out forming uneven bubbles that partly mimic insertion-deletion loops of mismatched DNA2. When the extruded hairpin exceeds 40-45 repeats, it can be bound by three or more MutSβ molecules, which are resistant to ATP-dependent dissociation. We envision that such MutSβ-CNG complexes recruit MutLγ endonuclease to nick DNA and initiate the repeat expansion process3,4. To develop drugs against the expansion diseases, we have identified lead compounds that prevent MutSβ binding to CNG repeats but not to mismatched DNA.
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Affiliation(s)
- Jun Li
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892
| | - Huaibin Wang
- Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892
| | - Wei Yang
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892
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4
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Gambelli A, Ferrando A, Boncristiani C, Schoeftner S. Regulation and function of R-loops at repetitive elements. Biochimie 2023; 214:141-155. [PMID: 37619810 DOI: 10.1016/j.biochi.2023.08.013] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2023] [Revised: 08/13/2023] [Accepted: 08/19/2023] [Indexed: 08/26/2023]
Abstract
R-loops are atypical, three-stranded nucleic acid structures that contain a stretch of RNA:DNA hybrids and an unpaired, single stranded DNA loop. R-loops are physiological relevant and can act as regulators of gene expression, chromatin structure, DNA damage repair and DNA replication. However, unscheduled and persistent R-loops are mutagenic and can mediate replication-transcription conflicts, leading to DNA damage and genome instability if left unchecked. Detailed transcriptome analysis unveiled that 85% of the human genome, including repetitive regions, hold transcriptional activity. This anticipates that R-loops management plays a central role for the regulation and integrity of genomes. This function is expected to have a particular relevance for repetitive sequences that make up to 75% of the human genome. Here, we review the impact of R-loops on the function and stability of repetitive regions such as centromeres, telomeres, rDNA arrays, transposable elements and triplet repeat expansions and discuss their relevance for associated pathological conditions.
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Affiliation(s)
- Alice Gambelli
- Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Via E. Weiss 2, 34127, Trieste, Italy
| | - Alessandro Ferrando
- Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Via E. Weiss 2, 34127, Trieste, Italy
| | - Chiara Boncristiani
- Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Via E. Weiss 2, 34127, Trieste, Italy
| | - Stefan Schoeftner
- Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Via E. Weiss 2, 34127, Trieste, Italy.
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5
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Gall-Duncan T, Luo J, Jurkovic CM, Fischer LA, Fujita K, Deshmukh AL, Harding RJ, Tran S, Mehkary M, Li V, Leib DE, Chen R, Tanaka H, Mason AG, Lévesque D, Khan M, Razzaghi M, Prasolava T, Lanni S, Sato N, Caron MC, Panigrahi GB, Wang P, Lau R, Castel AL, Masson JY, Tippett L, Turner C, Spies M, La Spada AR, Campos EI, Curtis MA, Boisvert FM, Faull RLM, Davidson BL, Nakamori M, Okazawa H, Wold MS, Pearson CE. Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability. Cell 2023; 186:4898-4919.e25. [PMID: 37827155 PMCID: PMC11209935 DOI: 10.1016/j.cell.2023.09.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 06/30/2023] [Accepted: 09/09/2023] [Indexed: 10/14/2023]
Abstract
Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.
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Affiliation(s)
- Terence Gall-Duncan
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Jennifer Luo
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | | | - Laura A Fischer
- Developmental Biology and Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Kyota Fujita
- Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
| | - Amit L Deshmukh
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Rachel J Harding
- Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada; Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Stephanie Tran
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Mustafa Mehkary
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Vanessa Li
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - David E Leib
- Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19146, USA
| | - Ran Chen
- Pediatrics, Division of Hematology and Oncology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Hikari Tanaka
- Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
| | - Amanda G Mason
- Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
| | - Dominique Lévesque
- Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Mahreen Khan
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Mortezaali Razzaghi
- Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Tanya Prasolava
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Stella Lanni
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Nozomu Sato
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Marie-Christine Caron
- CHU de Québec-Université Laval, Oncology Division, Molecular Biology, Medical Biochemistry, and Pathology, Laval University Cancer Research Center, Québec, QC, Canada
| | - Gagan B Panigrahi
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Peixiang Wang
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | - Rachel Lau
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada
| | | | - Jean-Yves Masson
- CHU de Québec-Université Laval, Oncology Division, Molecular Biology, Medical Biochemistry, and Pathology, Laval University Cancer Research Center, Québec, QC, Canada
| | - Lynette Tippett
- School of Psychology, University of Auckland, Auckland, New Zealand; University Research Centre for Brain Research, University of Auckland, Auckland, New Zealand
| | - Clinton Turner
- Anatomical Pathology, LabPlus, Auckland City Hospital, Auckland, New Zealand
| | - Maria Spies
- Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Albert R La Spada
- Pathology & Laboratory Medicine, Neurology, and Biological Chemistry, University of California, Irvine School of Medicine, Irvine, CA, USA; Neurobiology & Behavior, University of California, Irvine, Irvine, CA, USA; Center for Neurotherapeutics, University of California, Irvine, Irvine, CA 92697, USA
| | - Eric I Campos
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Maurice A Curtis
- University Research Centre for Brain Research, University of Auckland, Auckland, New Zealand; Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand
| | | | - Richard L M Faull
- University Research Centre for Brain Research, University of Auckland, Auckland, New Zealand; Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand
| | - Beverly L Davidson
- Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19146, USA
| | - Masayuki Nakamori
- Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Hitoshi Okazawa
- Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
| | - Marc S Wold
- Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Christopher E Pearson
- Genetics & Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada.
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6
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Abdi MH, Zamiri B, Pazuki G, Sardari S, Pearson CE. Pathogenic CANVAS-causing but not nonpathogenic RFC1 DNA/RNA repeat motifs form quadruplex or triplex structures. J Biol Chem 2023; 299:105202. [PMID: 37660923 PMCID: PMC10563062 DOI: 10.1016/j.jbc.2023.105202] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Revised: 08/22/2023] [Accepted: 08/25/2023] [Indexed: 09/05/2023] Open
Abstract
Biallelic expansions of various tandem repeat sequence motifs are possible in RFC1 (replication factor C subunit 1), encoding the DNA replication/repair protein RFC1, yet only certain repeat motifs cause cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). CANVAS presents enigmatic puzzles: The pathogenic path for CANVAS neither is known nor is it understood why some, but not all expanded, motifs are pathogenic. The most common pathogenic repeat is (AAGGG)n•(CCCTT)n, whereas (AAAAG)n•(CTTTT)n is the most common nonpathogenic motif. While both intronic motifs can be expanded and transcribed, only r(AAGGG)n is retained in the mutant RFC1 transcript. We show that only the pathogenic forms unusual nucleic acid structures. Specifically, DNA and RNA of the pathogenic d(AAGGG)4 and r(AAGGG)4 form G-quadruplexes in potassium solution. Nonpathogenic repeats did not form G-quadruplexes. Triple-stranded structures are formed by the pathogenic motifs but not by the nonpathogenic motifs. G- and C-richness of the pathogenic strands favor formation of G•G•G•G-tetrads and protonated C+-G Hoogsteen base pairings, involved in quadruplex and triplex structures, respectively, stabilized by increased hydrogen bonds and pi-stacking interactions relative to A-T Hoogsteen pairs that could form by the nonpathogenic motif. The ligand, TMPyP4, binds the pathogenic quadruplexes. Formation of quadruplexes and triplexes by pathogenic repeats supports toxic-DNA and toxic-RNA modes of pathogenesis at the RFC1 gene and the RFC1 transcript. Our findings with short repeats provide insights into the disease specificity of pathogenic repeat motif sequences and reveal nucleic acid structural features that may be pathogenically involved and targeted therapeutically.
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Affiliation(s)
- Mohammad Hossein Abdi
- Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran
| | - Bita Zamiri
- Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran.
| | - Gholamreza Pazuki
- Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran
| | - Soroush Sardari
- Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran
| | - Christopher E Pearson
- Program of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada.
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7
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O'Reilly D, Belgrad J, Ferguson C, Summers A, Sapp E, McHugh C, Mathews E, Boudi A, Buchwald J, Ly S, Moreno D, Furgal R, Luu E, Kennedy Z, Hariharan V, Monopoli K, Yang XW, Carroll J, DiFiglia M, Aronin N, Khvorova A. Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease. Mol Ther 2023; 31:1661-1674. [PMID: 37177784 PMCID: PMC10277892 DOI: 10.1016/j.ymthe.2023.05.006] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 03/10/2023] [Accepted: 05/08/2023] [Indexed: 05/15/2023] Open
Abstract
Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.
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Affiliation(s)
- Daniel O'Reilly
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Jillian Belgrad
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Chantal Ferguson
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Ashley Summers
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Ellen Sapp
- Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Cassandra McHugh
- Behavioral Neuroscience Program, Psychology Department, Western Washington University, Bellingham, WA 98225, USA
| | - Ella Mathews
- Behavioral Neuroscience Program, Psychology Department, Western Washington University, Bellingham, WA 98225, USA
| | - Adel Boudi
- Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Julianna Buchwald
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Socheata Ly
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Dimas Moreno
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Raymond Furgal
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Eric Luu
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Zachary Kennedy
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Vignesh Hariharan
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Kathryn Monopoli
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - X William Yang
- Center for Neurobehavioral Genetics, Jane and Terry Semel Institute of Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Jeffery Carroll
- Behavioral Neuroscience Program, Psychology Department, Western Washington University, Bellingham, WA 98225, USA; Department of Neurology, University of Washington, Seattle, WA 98104-2499, USA
| | - Marian DiFiglia
- Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Neil Aronin
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Anastasia Khvorova
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
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8
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Shibata T, Nakatani K. A small molecule binding to TGGAA pentanucleotide repeats that cause spinocerebellar ataxia type 31. Bioorg Med Chem Lett 2023; 79:129082. [PMID: 36414174 DOI: 10.1016/j.bmcl.2022.129082] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Revised: 11/07/2022] [Accepted: 11/16/2022] [Indexed: 11/21/2022]
Abstract
Spinocerebellar ataxia type 31 is an autosomal dominant neurodegenerative disease caused by aberrant insertion of d(TGGAA)n into the intron shared by brain expressed, associated with Nedd4 and thymidine kinase 2 genes in chromosome 16. We reported that a naphthyridine dimer derivative with amidated linker structure (ND-amide) bound to GGA/GGA motifs in hairpin structures of d(TGGAA)n. The binding of naphthyridine dimer derivatives to the GGA/GGA motif was sensitive to the linker structures. The amidation of the linker in naphthyridine dimer improved the binding property to the GGA/GGA motif as compared with non-amidated naphthyridine dimer.
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Affiliation(s)
- Tomonori Shibata
- Department of Regulatory Bioorganic Chemistry, SANKEN (The Institute of Scientific and Industrial Research), Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan.
| | - Kazuhiko Nakatani
- Department of Regulatory Bioorganic Chemistry, SANKEN (The Institute of Scientific and Industrial Research), Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan.
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9
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Heterogeneous migration routes of DNA triplet repeat slip-outs. BIOPHYSICAL REPORTS 2022; 2:None. [PMID: 36299495 PMCID: PMC9586884 DOI: 10.1016/j.bpr.2022.100070] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/07/2022] [Accepted: 08/08/2022] [Indexed: 12/02/2022]
Abstract
It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called "mobile" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)10 repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.
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10
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Deshmukh AL, Caron MC, Mohiuddin M, Lanni S, Panigrahi GB, Khan M, Engchuan W, Shum N, Faruqui A, Wang P, Yuen RKC, Nakamori M, Nakatani K, Masson JY, Pearson CE. FAN1 exo- not endo-nuclease pausing on disease-associated slipped-DNA repeats: A mechanism of repeat instability. Cell Rep 2021; 37:110078. [PMID: 34879276 DOI: 10.1016/j.celrep.2021.110078] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 07/02/2021] [Accepted: 11/09/2021] [Indexed: 12/19/2022] Open
Abstract
Ongoing inchworm-like CAG and CGG repeat expansions in brains, arising by aberrant processing of slipped DNAs, may drive Huntington's disease, fragile X syndrome, and autism. FAN1 nuclease modifies hyper-expansion rates by unknown means. We show that FAN1, through iterative cycles, binds, dimerizes, and cleaves slipped DNAs, yielding striking exo-nuclease pauses along slip-outs: 5'-C↓A↓GC↓A↓G-3' and 5'-C↓T↓G↓C↓T↓G-3'. CAG excision is slower than CTG and requires intra-strand A·A and T·T mismatches. Fully paired hairpins arrested excision, whereas disease-delaying CAA interruptions further slowed excision. Endo-nucleolytic cleavage is insensitive to slip-outs. Rare FAN1 variants are found in individuals with autism with CGG/CCG expansions, and CGG/CCG slip-outs show exo-nuclease pauses. The slip-out-specific ligand, naphthyridine-azaquinolone, which induces contractions of expanded repeats in vivo, requires FAN1 for its effect, and protects slip-outs from FAN1 exo-, but not endo-, nucleolytic digestion. FAN1's inchworm pausing of slip-out excision rates is well suited to modify inchworm expansion rates, which modify disease onset and progression.
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Affiliation(s)
- Amit Laxmikant Deshmukh
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Marie-Christine Caron
- Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Division, Québec City, QC G1R 3S3, Canada; Department of Molecular Biology, Medical Biochemistry, and Pathology, Laval University Cancer Research Center, Québec City, QC G1R 3S3, Canada
| | - Mohiuddin Mohiuddin
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Stella Lanni
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Gagan B Panigrahi
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Mahreen Khan
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Program of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Worrawat Engchuan
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Natalie Shum
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Program of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Aisha Faruqui
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Program of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Peixiang Wang
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada
| | - Ryan K C Yuen
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Program of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Masayuki Nakamori
- Department of Neurology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
| | - Kazuhiko Nakatani
- Department of Regulatory Bioorganic Chemistry, the Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan
| | - Jean-Yves Masson
- Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Division, Québec City, QC G1R 3S3, Canada; Department of Molecular Biology, Medical Biochemistry, and Pathology, Laval University Cancer Research Center, Québec City, QC G1R 3S3, Canada
| | - Christopher E Pearson
- Program of Genetics & Genome Biology, The Hospital for Sick Children, PGCRL, Toronto, Canada, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Program of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
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11
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Abstract
At fifteen different genomic locations, the expansion of a CAG/CTG repeat causes a neurodegenerative or neuromuscular disease, the most common being Huntington's disease and myotonic dystrophy type 1. These disorders are characterized by germline and somatic instability of the causative CAG/CTG repeat mutations. Repeat lengthening, or expansion, in the germline leads to an earlier age of onset or more severe symptoms in the next generation. In somatic cells, repeat expansion is thought to precipitate the rate of disease. The mechanisms underlying repeat instability are not well understood. Here we review the mammalian model systems that have been used to study CAG/CTG repeat instability, and the modifiers identified in these systems. Mouse models have demonstrated prominent roles for proteins in the mismatch repair pathway as critical drivers of CAG/CTG instability, which is also suggested by recent genome-wide association studies in humans. We draw attention to a network of connections between modifiers identified across several systems that might indicate pathway crosstalk in the context of repeat instability, and which could provide hypotheses for further validation or discovery. Overall, the data indicate that repeat dynamics might be modulated by altering the levels of DNA metabolic proteins, their regulation, their interaction with chromatin, or by direct perturbation of the repeat tract. Applying novel methodologies and technologies to this exciting area of research will be needed to gain deeper mechanistic insight that can be harnessed for therapies aimed at preventing repeat expansion or promoting repeat contraction.
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Affiliation(s)
- Vanessa C. Wheeler
- Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA,Department of Neurology, Harvard Medical School, Boston, MA, USA,Correspondence to: Vanessa C. Wheeler, Center for Genomic Medicine, Massachusetts Hospital, Boston MAA 02115, USA. E-mail: . and Vincent Dion, UK Dementia Research Institute at Cardiff University, Hadyn Ellis Building, Maindy Road, CF24 4HQ Cardiff, UK. E-mail:
| | - Vincent Dion
- UK Dementia Research Institute at Cardiff University, Hadyn Ellis Building, Maindy Road, Cardiff, UK,Correspondence to: Vanessa C. Wheeler, Center for Genomic Medicine, Massachusetts Hospital, Boston MAA 02115, USA. E-mail: . and Vincent Dion, UK Dementia Research Institute at Cardiff University, Hadyn Ellis Building, Maindy Road, CF24 4HQ Cardiff, UK. E-mail:
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12
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Porro A, Mohiuddin M, Zurfluh C, Spegg V, Dai J, Iehl F, Ropars V, Collotta G, Fishwick KM, Mozaffari NL, Guérois R, Jiricny J, Altmeyer M, Charbonnier JB, Pearson CE, Sartori AA. FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats. SCIENCE ADVANCES 2021; 7:7/31/eabf7906. [PMID: 34330701 PMCID: PMC8324060 DOI: 10.1126/sciadv.abf7906] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Accepted: 06/15/2021] [Indexed: 05/05/2023]
Abstract
FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.
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Affiliation(s)
- Antonio Porro
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Mohiuddin Mohiuddin
- Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
| | - Christina Zurfluh
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Vincent Spegg
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Jingqi Dai
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Florence Iehl
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Virginie Ropars
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Giulio Collotta
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Keri M Fishwick
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Nour L Mozaffari
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Raphaël Guérois
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Josef Jiricny
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Matthias Altmeyer
- Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland
| | - Jean-Baptiste Charbonnier
- Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France
| | - Christopher E Pearson
- Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
- Program of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Alessandro A Sartori
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
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13
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Oura S, Noda T, Morimura N, Hitoshi S, Nishimasu H, Nagai Y, Nureki O, Ikawa M. Precise CAG repeat contraction in a Huntington's Disease mouse model is enabled by gene editing with SpCas9-NG. Commun Biol 2021; 4:771. [PMID: 34163001 PMCID: PMC8222283 DOI: 10.1038/s42003-021-02304-w] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2020] [Accepted: 06/03/2021] [Indexed: 12/22/2022] Open
Abstract
The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a research hotspot in gene therapy. However, the widely used Streptococcus pyogenes Cas9 (WT-SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting targetable disease mutations. To address this issue, we recently reported an engineered SpCas9 nuclease variant (SpCas9-NG) recognizing NGN PAMs. Here, as a feasibility study, we report SpCas9-NG-mediated repair of the abnormally expanded CAG repeat tract in Huntington's disease (HD). By targeting the boundary of CAG repeats with SpCas9-NG, we precisely contracted the repeat tracts in HD-mouse-derived embryonic stem (ES) cells. Further, we confirmed the recovery of phenotypic abnormalities in differentiated neurons and animals produced from repaired ES cells. Our study shows that SpCas9-NG can be a powerful tool for repairing abnormally expanded CAG repeats as well as other disease mutations that are difficult to access with WT-SpCas9.
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Affiliation(s)
- Seiya Oura
- Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Taichi Noda
- Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- Division of Reproductive Biology, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan
| | - Naoko Morimura
- Department of Integrative Physiology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Seiji Hitoshi
- Department of Integrative Physiology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Hiroshi Nishimasu
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
- Department of Structural Biology, Research center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan
| | - Yoshitaka Nagai
- Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan
- Department of Neurotherapeutics, Osaka University Graduate School of Medicine, Osaka, Japan
- Department of Neurology, Kindai University Faculty of Medicine, Osaka, Japan
| | - Osamu Nureki
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
| | - Masahito Ikawa
- Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
- Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
- Laboratory of Reproductive Systems Biology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
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14
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Dohno C, Hagihara M, Binti Mohd Zaifuddin N, Nihei M, Saito K, Nakatani K. Small molecule-induced trinucleotide repeat contractions during in vitro DNA synthesis. Chem Commun (Camb) 2021; 57:3235-3238. [PMID: 33646236 DOI: 10.1039/d1cc00349f] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
We demonstrated that a synthetic ligand NA, which selectively binds to a 5'-CAG-3'/5'-CAG-3' triad, induced repeat contractions during DNA polymerase-mediated primer extension through the CAG repeat template. A thorough capillary electrophoresis and sequencing analysis revealed that the d(CAG)20 template gave shortened nascent strands mainly containing 3-6 CTG units in the presence of NA.
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Affiliation(s)
- Chikara Dohno
- The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
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15
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Young SJ, West SC. Coordinated roles of SLX4 and MutSβ in DNA repair and the maintenance of genome stability. Crit Rev Biochem Mol Biol 2021; 56:157-177. [PMID: 33596761 PMCID: PMC7610648 DOI: 10.1080/10409238.2021.1881433] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Revised: 01/06/2021] [Accepted: 01/22/2021] [Indexed: 12/14/2022]
Abstract
SLX4 provides a molecular scaffold for the assembly of multiple protein complexes required for the maintenance of genome stability. It is involved in the repair of DNA crosslinks, the resolution of recombination intermediates, the response to replication stress and the maintenance of telomere length. To carry out these diverse functions, SLX4 interacts with three structure-selective endonucleases, MUS81-EME1, SLX1 and XPF-ERCC1, as well as the telomere binding proteins TRF2, RTEL1 and SLX4IP. Recently, SLX4 was shown to interact with MutSβ, a heterodimeric protein involved in DNA mismatch repair, trinucleotide repeat instability, crosslink repair and recombination. Importantly, MutSβ promotes the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease. The colocalization and specific interaction of MutSβ with SLX4, together with their apparently overlapping functions, are suggestive of a common role in reactions that promote DNA maintenance and genome stability. This review will focus on the role of SLX4 in DNA repair, the interplay between MutSβ and SLX4, and detail how they cooperate to promote recombinational repair and DNA crosslink repair. Furthermore, we speculate that MutSβ and SLX4 may provide an alternative cellular mechanism that modulates trinucleotide instability.
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Affiliation(s)
- Sarah J Young
- DNA Recombination and Repair Laboratory, The Francis Crick Institute, London, UK
| | - Stephen C West
- DNA Recombination and Repair Laboratory, The Francis Crick Institute, London, UK
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16
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Abstract
DNA mismatch repair (MMR) is a highly conserved genome stabilizing pathway that corrects DNA replication errors, limits chromosomal rearrangements, and mediates the cellular response to many types of DNA damage. Counterintuitively, MMR is also involved in the generation of mutations, as evidenced by its role in causing somatic triplet repeat expansion in Huntington’s disease (HD) and other neurodegenerative disorders. In this review, we discuss the current state of mechanistic knowledge of MMR and review the roles of key enzymes in this pathway. We also present the evidence for mutagenic function of MMR in CAG repeat expansion and consider mechanistic hypotheses that have been proposed. Understanding the role of MMR in CAG expansion may shed light on potential avenues for therapeutic intervention in HD.
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Affiliation(s)
- Ravi R Iyer
- CHDI Management/CHDI Foundation, Princeton, NJ, USA
| | - Anna Pluciennik
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
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17
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Deshmukh AL, Porro A, Mohiuddin M, Lanni S, Panigrahi GB, Caron MC, Masson JY, Sartori AA, Pearson CE. FAN1, a DNA Repair Nuclease, as a Modifier of Repeat Expansion Disorders. J Huntingtons Dis 2021; 10:95-122. [PMID: 33579867 PMCID: PMC7990447 DOI: 10.3233/jhd-200448] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
FAN1 encodes a DNA repair nuclease. Genetic deficiencies, copy number variants, and single nucleotide variants of FAN1 have been linked to karyomegalic interstitial nephritis, 15q13.3 microdeletion/microduplication syndrome (autism, schizophrenia, and epilepsy), cancer, and most recently repeat expansion diseases. For seven CAG repeat expansion diseases (Huntington's disease (HD) and certain spinocerebellar ataxias), modification of age of onset is linked to variants of specific DNA repair proteins. FAN1 variants are the strongest modifiers. Non-coding disease-delaying FAN1 variants and coding disease-hastening variants (p.R507H and p.R377W) are known, where the former may lead to increased FAN1 levels and the latter have unknown effects upon FAN1 functions. Current thoughts are that ongoing repeat expansions in disease-vulnerable tissues, as individuals age, promote disease onset. Fan1 is required to suppress against high levels of ongoing somatic CAG and CGG repeat expansions in tissues of HD and FMR1 transgenic mice respectively, in addition to participating in DNA interstrand crosslink repair. FAN1 is also a modifier of autism, schizophrenia, and epilepsy. Coupled with the association of these diseases with repeat expansions, this suggests a common mechanism, by which FAN1 modifies repeat diseases. Yet how any of the FAN1 variants modify disease is unknown. Here, we review FAN1 variants, associated clinical effects, protein structure, and the enzyme's attributed functional roles. We highlight how variants may alter its activities in DNA damage response and/or repeat instability. A thorough awareness of the FAN1 gene and FAN1 protein functions will reveal if and how it may be targeted for clinical benefit.
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Affiliation(s)
- Amit L. Deshmukh
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Antonio Porro
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
| | - Mohiuddin Mohiuddin
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Stella Lanni
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Gagan B. Panigrahi
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Marie-Christine Caron
- Department of Molecular Biology, Medical Biochemistry and Pathology; Laval University Cancer Research Center, Québec City, Quebec, Canada
- Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Division, Québec City, Quebec, Canada
| | - Jean-Yves Masson
- Department of Molecular Biology, Medical Biochemistry and Pathology; Laval University Cancer Research Center, Québec City, Quebec, Canada
- Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Division, Québec City, Quebec, Canada
| | | | - Christopher E. Pearson
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
- University of Toronto, Program of Molecular Genetics, Toronto, Ontario, Canada
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18
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Alternative DNA Structures In Vivo: Molecular Evidence and Remaining Questions. Microbiol Mol Biol Rev 2020; 85:85/1/e00110-20. [PMID: 33361270 DOI: 10.1128/mmbr.00110-20] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Duplex DNA naturally folds into a right-handed double helix in physiological conditions. Some sequences of unusual base composition may nevertheless form alternative structures, as was shown for many repeated sequences in vitro However, evidence for the formation of noncanonical structures in living cells is difficult to gather. It mainly relies on genetic assays demonstrating their function in vivo or through genetic instability reflecting particular properties of such structures. Efforts were made to reveal their existence directly in a living cell, mainly by generating antibodies specific to secondary structures or using chemical ligands selected for their affinity to these structures. Among secondary structure-forming DNAs are G-quadruplexes, human fragile sites containing minisatellites, AT-rich regions, inverted repeats able to form cruciform structures, hairpin-forming CAG/CTG triplet repeats, and triple helices formed by homopurine-homopyrimidine GAA/TTC trinucleotide repeats. Many of these alternative structures are involved in human pathologies, such as neurological or developmental disorders, as in the case of trinucleotide repeats, or cancers triggered by translocations linked to fragile sites. This review will discuss and highlight evidence supporting the formation of alternative DNA structures in vivo and will emphasize the role of the mismatch repair machinery in binding mispaired DNA duplexes, triggering genetic instability.
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19
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Young SJ, Sebald M, Shah Punatar R, Larin M, Masino L, Rodrigo-Brenni MC, Liang CC, West SC. MutSβ Stimulates Holliday Junction Resolution by the SMX Complex. Cell Rep 2020; 33:108289. [PMID: 33086055 DOI: 10.1016/j.celrep.2020.108289] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Revised: 09/02/2020] [Accepted: 09/29/2020] [Indexed: 12/20/2022] Open
Abstract
MutSα and MutSβ play important roles in DNA mismatch repair and are linked to inheritable cancers and degenerative disorders. Here, we show that MSH2 and MSH3, the two components of MutSβ, bind SLX4 protein, a scaffold for the assembly of the SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) trinuclease complex. SMX promotes the resolution of Holliday junctions (HJs), which are intermediates in homologous recombinational repair. We find that MutSβ binds HJs and stimulates their resolution by SLX1-SLX4 or SMX in reactions dependent upon direct interactions between MutSβ and SLX4. In contrast, MutSα does not stimulate HJ resolution. MSH3-depleted cells exhibit reduced sister chromatid exchanges and elevated levels of homologous recombination ultrafine bridges (HR-UFBs) at mitosis, consistent with defects in the processing of recombination intermediates. These results demonstrate a role for MutSβ in addition to its established role in the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease.
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Affiliation(s)
- Sarah J Young
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Marie Sebald
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | | | - Meghan Larin
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Laura Masino
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | | | - Chih-Chao Liang
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Stephen C West
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
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20
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Gadgil RY, Romer EJ, Goodman CC, Rider SD, Damewood FJ, Barthelemy JR, Shin-Ya K, Hanenberg H, Leffak M. Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication. J Biol Chem 2020; 295:15378-15397. [PMID: 32873711 DOI: 10.1074/jbc.ra120.013495] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2020] [Revised: 08/23/2020] [Indexed: 12/12/2022] Open
Abstract
Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG) n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG) n chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.
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Affiliation(s)
- Rujuta Yashodhan Gadgil
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA
| | - Eric J Romer
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA
| | - Caitlin C Goodman
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA
| | - S Dean Rider
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA
| | - French J Damewood
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA
| | - Joanna R Barthelemy
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA
| | - Kazuo Shin-Ya
- Biomedical Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan
| | - Helmut Hanenberg
- Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University, Düsseldorf, Germany; Department of Pediatrics III, University Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Michael Leffak
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA.
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21
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Tabrizi SJ, Flower MD, Ross CA, Wild EJ. Huntington disease: new insights into molecular pathogenesis and therapeutic opportunities. Nat Rev Neurol 2020; 16:529-546. [PMID: 32796930 DOI: 10.1038/s41582-020-0389-4] [Citation(s) in RCA: 313] [Impact Index Per Article: 62.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/29/2020] [Indexed: 12/11/2022]
Abstract
Huntington disease (HD) is a neurodegenerative disease caused by CAG repeat expansion in the huntingtin gene (HTT) and involves a complex web of pathogenic mechanisms. Mutant HTT (mHTT) disrupts transcription, interferes with immune and mitochondrial function, and is aberrantly modified post-translationally. Evidence suggests that the mHTT RNA is toxic, and at the DNA level, somatic CAG repeat expansion in vulnerable cells influences the disease course. Genome-wide association studies have identified DNA repair pathways as modifiers of somatic instability and disease course in HD and other repeat expansion diseases. In animal models of HD, nucleocytoplasmic transport is disrupted and its restoration is neuroprotective. Novel cerebrospinal fluid (CSF) and plasma biomarkers are among the earliest detectable changes in individuals with premanifest HD and have the sensitivity to detect therapeutic benefit. Therapeutically, the first human trial of an HTT-lowering antisense oligonucleotide successfully, and safely, reduced the CSF concentration of mHTT in individuals with HD. A larger trial, powered to detect clinical efficacy, is underway, along with trials of other HTT-lowering approaches. In this Review, we discuss new insights into the molecular pathogenesis of HD and future therapeutic strategies, including the modulation of DNA repair and targeting the DNA mutation itself.
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Affiliation(s)
- Sarah J Tabrizi
- Huntington's Disease Centre, University College London, London, UK. .,Department of Neurodegenerative Disease, Queen Square Institute of Neurology, University College London, London, UK. .,UK Dementia Research Institute, University College London, London, UK.
| | - Michael D Flower
- Huntington's Disease Centre, University College London, London, UK.,Department of Neurodegenerative Disease, Queen Square Institute of Neurology, University College London, London, UK.,UK Dementia Research Institute, University College London, London, UK
| | - Christopher A Ross
- Departments of Neurology, Neuroscience and Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Edward J Wild
- Huntington's Disease Centre, University College London, London, UK.,Department of Neurodegenerative Disease, Queen Square Institute of Neurology, University College London, London, UK
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22
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Mosbach V, Viterbo D, Descorps-Declère S, Poggi L, Vaysse-Zinkhöfer W, Richard GF. Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. PLoS Genet 2020; 16:e1008924. [PMID: 32673314 PMCID: PMC7413560 DOI: 10.1371/journal.pgen.1008924] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Revised: 08/07/2020] [Accepted: 06/10/2020] [Indexed: 12/26/2022] Open
Abstract
Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome. With the discovery of highly specific DNA endonucleases such as TALEN and CRISPR-Cas systems, gene editing has become an attractive approach to address genetic disorders. Myotonic dystrophy type 1 (Steinert disease) is due to a large expansion of a CTG trinucleotide repeat in the DMPK gene. At the present time, despite numerous therapeutic attempts, this dramatic neurodegenerative disorder still has no cure. In the present work, we tried to use the Cas9 endonuclease to induce a double-strand break within the expanded CTG repeat of the DMPK gene integrated in the yeast genome. Surprisingly, this break induced chromosomal deletions around the repeat tract. These deletions were local and involved non-homologous joining of the two DNA ends, or more extensive involving homologous recombination between repeated elements upstream and downstream the break. Using yeast genetics, we investigated the genetic requirements for these deletions and found that the triplet repeat tract altered the capacity of the repair machinery to faithfully repair the double-strand break. These results have implications for future gene therapy approaches in human patients.
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Affiliation(s)
| | | | - Stéphane Descorps-Declère
- Institut Pasteur, CNRS, UMR3525, Paris, France
- Institut Pasteur, Center of Bioinformatics, Biostatistics and Integrative Biology (C3BI), Paris, France
| | - Lucie Poggi
- Institut Pasteur, CNRS, UMR3525, Paris, France
- Sorbonne Universités, Collège doctoral, Paris, France
| | - Wilhelm Vaysse-Zinkhöfer
- Institut Pasteur, CNRS, UMR3525, Paris, France
- Sorbonne Universités, Collège doctoral, Paris, France
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23
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Xu P, Pan F, Roland C, Sagui C, Weninger K. Dynamics of strand slippage in DNA hairpins formed by CAG repeats: roles of sequence parity and trinucleotide interrupts. Nucleic Acids Res 2020; 48:2232-2245. [PMID: 31974547 PMCID: PMC7049705 DOI: 10.1093/nar/gkaa036] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Revised: 12/11/2019] [Accepted: 01/15/2020] [Indexed: 01/08/2023] Open
Abstract
DNA trinucleotide repeats (TRs) can exhibit dynamic expansions by integer numbers of trinucleotides that lead to neurodegenerative disorders. Strand slipped hairpins during DNA replication, repair and/or recombination may contribute to TR expansion. Here, we combine single-molecule FRET experiments and molecular dynamics studies to elucidate slipping dynamics and conformations of (CAG)n TR hairpins. We directly resolve slipping by predominantly two CAG units. The slipping kinetics depends on the even/odd repeat parity. The populated states suggest greater stability for 5′-AGCA-3′ tetraloops, compared with alternative 5′-CAG-3′ triloops. To accommodate the tetraloop, even(odd)-numbered repeats have an even(odd) number of hanging bases in the hairpin stem. In particular, a paired-end tetraloop (no hanging TR) is stable in (CAG)n = even, but such situation cannot occur in (CAG)n = odd, where the hairpin is “frustrated’’ and slips back and forth between states with one TR hanging at the 5′ or 3′ end. Trinucleotide interrupts in the repeating CAG pattern associated with altered disease phenotypes select for specific conformers with favorable loop sequences. Molecular dynamics provide atomic-level insight into the loop configurations. Reducing strand slipping in TR hairpins by sequence interruptions at the loop suggests disease-associated variations impact expansion mechanisms at the level of slipped hairpins.
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Affiliation(s)
- Pengning Xu
- Department of Physics, North Carolina State University, Raleigh, NC 27695-8202, USA
| | - Feng Pan
- Department of Physics, North Carolina State University, Raleigh, NC 27695-8202, USA
| | - Christopher Roland
- Department of Physics, North Carolina State University, Raleigh, NC 27695-8202, USA
| | - Celeste Sagui
- Department of Physics, North Carolina State University, Raleigh, NC 27695-8202, USA
| | - Keith Weninger
- Department of Physics, North Carolina State University, Raleigh, NC 27695-8202, USA
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24
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Affiliation(s)
- Michael D Flower
- University College London (UCL) Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology and Dementia Research Centre at UCL, London, UK
| | - Sarah J Tabrizi
- University College London (UCL) Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology and Dementia Research Centre at UCL, London, UK.
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25
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Khristich AN, Mirkin SM. On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability. J Biol Chem 2020; 295:4134-4170. [PMID: 32060097 PMCID: PMC7105313 DOI: 10.1074/jbc.rev119.007678] [Citation(s) in RCA: 188] [Impact Index Per Article: 37.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability. Next, we describe the consequences of the presence of long structure-forming repeats at the molecular level: somatic and intergenerational instability, fragility, and repeat-induced mutagenesis. We discuss the reasons for gender bias in intergenerational repeat instability and the tissue specificity of somatic repeat instability. We also review the known pathways in which DNA replication, transcription, DNA repair, and chromatin state interact and thereby promote repeat instability. We then discuss possible reasons for the persistence of disease-causing DNA repeats in the genome. We describe evidence suggesting that these repeats are a payoff for the advantages of having abundant simple-sequence repeats for eukaryotic genome function and evolvability. Finally, we discuss two unresolved fundamental questions: (i) why does repeat behavior differ between model systems and human pedigrees, and (ii) can we use current knowledge on repeat instability mechanisms to cure repeat expansion diseases?
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Affiliation(s)
| | - Sergei M Mirkin
- Department of Biology, Tufts University, Medford, Massachusetts 02155.
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26
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Human MutLγ, the MLH1-MLH3 heterodimer, is an endonuclease that promotes DNA expansion. Proc Natl Acad Sci U S A 2020; 117:3535-3542. [PMID: 32015124 PMCID: PMC7035508 DOI: 10.1073/pnas.1914718117] [Citation(s) in RCA: 56] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
MutL proteins are ubiquitous and play important roles in DNA metabolism. MutLγ (MLH1-MLH3 heterodimer) is a poorly understood member of the eukaryotic family of MutL proteins that has been implicated in triplet repeat expansion, but its action in this deleterious process has remained unknown. In humans, triplet repeat expansion is the molecular basis for ∼40 neurological disorders. In addition to MutLγ, triplet repeat expansion involves the mismatch recognition factor MutSβ (MSH2-MSH3 heterodimer). We show here that human MutLγ is an endonuclease that nicks DNA. Strikingly, incision of covalently closed, relaxed loop-containing DNA by human MutLγ is promoted by MutSβ and targeted to the strand opposite the loop. The resulting strand break licenses downstream events that lead to a DNA expansion event in human cell extracts. Our data imply that the mammalian MutLγ is a unique endonuclease that can initiate triplet repeat DNA expansions.
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27
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Nakamori M, Panigrahi GB, Lanni S, Gall-Duncan T, Hayakawa H, Tanaka H, Luo J, Otabe T, Li J, Sakata A, Caron MC, Joshi N, Prasolava T, Chiang K, Masson JY, Wold MS, Wang X, Lee MYWT, Huddleston J, Munson KM, Davidson S, Layeghifard M, Edward LM, Gallon R, Santibanez-Koref M, Murata A, Takahashi MP, Eichler EE, Shlien A, Nakatani K, Mochizuki H, Pearson CE. A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo. Nat Genet 2020; 52:146-159. [PMID: 32060489 PMCID: PMC7043212 DOI: 10.1038/s41588-019-0575-8] [Citation(s) in RCA: 95] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2018] [Accepted: 12/19/2019] [Indexed: 01/07/2023]
Abstract
In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSβ. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.
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Affiliation(s)
- Masayuki Nakamori
- Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Gagan B Panigrahi
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Stella Lanni
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Terence Gall-Duncan
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Hideki Hayakawa
- Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Hana Tanaka
- Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Jennifer Luo
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Takahiro Otabe
- Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan
| | - Jinxing Li
- Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan
| | - Akihiro Sakata
- Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan
| | - Marie-Christine Caron
- Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Division, Quebec, Quebec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec, Quebec, Canada
| | - Niraj Joshi
- Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Division, Quebec, Quebec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec, Quebec, Canada
| | - Tanya Prasolava
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Karen Chiang
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Jean-Yves Masson
- Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Division, Quebec, Quebec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec, Quebec, Canada
| | - Marc S Wold
- Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Xiaoxiao Wang
- Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, USA
| | - Marietta Y W T Lee
- Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, USA
| | - John Huddleston
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
- Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA
| | - Katherine M Munson
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
| | - Scott Davidson
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Mehdi Layeghifard
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Lisa-Monique Edward
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Richard Gallon
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK
| | | | - Asako Murata
- Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan
| | - Masanori P Takahashi
- Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Evan E Eichler
- Department of Genome Sciences, University of Washington, Seattle, WA, USA
- Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA
| | - Adam Shlien
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada
| | - Kazuhiko Nakatani
- Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan
| | - Hideki Mochizuki
- Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Christopher E Pearson
- Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada.
- Program of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
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28
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Zheng J, Xu H, Cao H. A Long Polymorphic GT Microsatellite within a Gene Promoter Mediates Non-Imprinted Allele-Specific DNA Methylation of a CpG Island in a Goldfish Inter-Strain Hybrid. Int J Mol Sci 2019; 20:ijms20163923. [PMID: 31409051 PMCID: PMC6721770 DOI: 10.3390/ijms20163923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2019] [Revised: 08/01/2019] [Accepted: 08/06/2019] [Indexed: 11/26/2022] Open
Abstract
It is now widely accepted that allele-specific DNA methylation (ASM) commonly occurs at non-imprinted loci. Most of the non-imprinted ASM regions observed both within and outside of the CpG island show a strong correlation with DNA polymorphisms. However, what polymorphic cis-acting elements mediate non-imprinted ASM of the CpG island remains unclear. In this study, we investigated the impact of polymorphic GT microsatellites within the gene promoter on non-imprinted ASM of the local CpG island in goldfish. We generated various goldfish heterozygotes, in which the length of GT microsatellites or some non-repetitive sequences in the promoter of no tail alleles was different. By examining the methylation status of the downstream CpG island in these heterozygotes, we found that polymorphisms of a long GT microsatellite can lead to the ASM of the downstream CpG island during oogenesis and embryogenesis, polymorphisms of short GT microsatellites and non-repetitive sequences in the promoter exhibited no significant effect on the methylation of the CpG island. We also observed that the ASM of the CpG island was associated with allele-specific expression in heterozygous embryos. These results suggest that a long polymorphic GT microsatellite within a gene promoter mediates non-imprinted ASM of the local CpG island in a goldfish inter-strain hybrid.
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Affiliation(s)
- Jianbo Zheng
- Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China.
- College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
| | - Haomang Xu
- College of Life Sciences, Zhejiang University, Hangzhou 310058, China
| | - Huiwen Cao
- College of Life Sciences, Zhejiang University, Hangzhou 310058, China
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29
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Raaijmakers RHL, Ripken L, Ausems CRM, Wansink DG. CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities. Int J Mol Sci 2019; 20:ijms20153689. [PMID: 31357652 PMCID: PMC6696057 DOI: 10.3390/ijms20153689] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 07/24/2019] [Accepted: 07/25/2019] [Indexed: 02/06/2023] Open
Abstract
CRISPR/Cas technology holds promise for the development of therapies to treat inherited diseases. Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder with a variable multisystemic character for which no cure is yet available. Here, we review CRISPR/Cas-mediated approaches that target the unstable (CTG•CAG)n repeat in the DMPK/DM1-AS gene pair, the autosomal dominant mutation that causes DM1. Expansion of the repeat results in a complex constellation of toxicity at the DNA level, an altered transcriptome and a disturbed proteome. To restore cellular homeostasis and ameliorate DM1 disease symptoms, CRISPR/Cas approaches were directed at the causative mutation in the DNA and the RNA. Specifically, the triplet repeat has been excised from the genome by several laboratories via dual CRISPR/Cas9 cleavage, while one group prevented transcription of the (CTG)n repeat through homology-directed insertion of a polyadenylation signal in DMPK. Independently, catalytically deficient Cas9 (dCas9) was recruited to the (CTG)n repeat to block progression of RNA polymerase II and a dCas9-RNase fusion was shown to degrade expanded (CUG)n RNA. We compare these promising developments in DM1 with those in other microsatellite instability diseases. Finally, we look at hurdles that must be taken to make CRISPR/Cas-mediated editing a therapeutic reality in patients.
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Affiliation(s)
- Renée H L Raaijmakers
- Department of Cell Biology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6525 GA Nijmegen, The Netherlands
- Department of Human Genetics, Radboud University Medical Center, Donders Institute for Brain Cognition and Behavior, 6525 GA Nijmegen, The Netherlands
| | - Lise Ripken
- Department of Cell Biology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6525 GA Nijmegen, The Netherlands
| | - C Rosanne M Ausems
- Department of Cell Biology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6525 GA Nijmegen, The Netherlands
- Department of Human Genetics, Radboud University Medical Center, Donders Institute for Brain Cognition and Behavior, 6525 GA Nijmegen, The Netherlands
| | - Derick G Wansink
- Department of Cell Biology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6525 GA Nijmegen, The Netherlands.
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30
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Loureiro JR, Oliveira CL, Mota C, Castro AF, Costa C, Loureiro JL, Coutinho P, Martins S, Sequeiros J, Silveira I. Mutational mechanism for DAB1 (ATTTC) n insertion in SCA37: ATTTT repeat lengthening and nucleotide substitution. Hum Mutat 2019; 40:404-412. [PMID: 30588707 DOI: 10.1002/humu.23704] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Revised: 11/28/2018] [Accepted: 12/22/2018] [Indexed: 12/20/2022]
Abstract
Dynamic mutations by microsatellite instability are the molecular basis of a growing number of neuromuscular and neurodegenerative diseases. Repetitive stretches in the human genome may drive pathogenicity, either by expansion above a given threshold, or by insertion of abnormal tracts in nonpathogenic polymorphic repetitive regions, as is the case in spinocerebellar ataxia type 37 (SCA37). We have recently established that this neurodegenerative disease is caused by an (ATTTC)n insertion within an (ATTTT)n in a noncoding region of DAB1. We now investigated the mutational mechanism that originated the (ATTTC)n insertion within an ancestral (ATTTT)n . Approximately 3% of nonpathogenic (ATTTT)n alleles are interspersed by AT-rich motifs, contrarily to mutant alleles that are composed of pure (ATTTT)n and (ATTTC)n stretches. Haplotype studies in unaffected chromosomes suggested that the primary mutational mechanism, leading to the (ATTTC)n insertion, was likely one or more T>C substitutions in an (ATTTT)n pure allele of approximately 200 repeats. Then, the (ATTTC)n expanded in size, originating a deleterious allele in DAB1 that leads to SCA37. This is likely the mutational mechanism in three similar (TTTCA)n insertions responsible for familial myoclonic epilepsy. Because (ATTTT)n tracts are frequent in the human genome, many loci could be at risk for this mutational process.
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Affiliation(s)
- Joana R Loureiro
- Genetics of Cognitive Dysfunction Laboratory, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.,IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal.,ICBAS, Universidade do Porto, Porto, Portugal
| | - Cláudia L Oliveira
- Genetics of Cognitive Dysfunction Laboratory, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.,IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal
| | - Carolina Mota
- Genetics of Cognitive Dysfunction Laboratory, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.,IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal
| | - Ana F Castro
- Genetics of Cognitive Dysfunction Laboratory, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.,IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal
| | - Cristina Costa
- Department of Neurology, Hospital Prof. Doutor Fernando Fonseca, Amadora, Portugal
| | - José L Loureiro
- IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal.,UnIGENe, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.,Department of Neurology, Hospital São Sebastião, Feira, Portugal
| | - Paula Coutinho
- IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal.,UnIGENe, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
| | - Sandra Martins
- Population Genetics & Evolution, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.,IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal
| | - Jorge Sequeiros
- IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal.,ICBAS, Universidade do Porto, Porto, Portugal.,UnIGENe, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
| | - Isabel Silveira
- Genetics of Cognitive Dysfunction Laboratory, i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.,IBMC- Institute for Molecular and Cell Biology, Universidade do Porto, Porto, Portugal
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31
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Mitchell ML, Leveille MP, Solecki RS, Tran T, Cannon B. Sequence-Dependent Effects of Monovalent Cations on the Structural Dynamics of Trinucleotide-Repeat DNA Hairpins. J Phys Chem B 2018; 122:11841-11851. [PMID: 30441902 DOI: 10.1021/acs.jpcb.8b07994] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Repetitive trinucleotide DNA sequences at specific genetic loci are associated with numerous hereditary, neurodegenerative diseases. The propensity of single-stranded domains containing these sequences to form secondary structure via extensive self-complementarity disrupts normal DNA processing to create genetic instabilities. To investigate these intrastrand structural dynamics, a DNA hairpin system was devised for single-molecule fluorescence study of the folding kinetics and energetics for secondary structure formation between two interacting, repetitive domains with specific numbers of the same trinucleotide motif (CXG), where X = T or A. Single-molecule fluorescence resonance energy transfer (smFRET) data show discrete conformational transitions between unstructured and closed hairpin states. The lifetimes of the closed hairpin states correlate with the number of repeats, with (CTG) N/(CTG) N domains maintaining longer-lived, closed states than equivalent-sized (CAG) N/(CAG) N domains. NaCl promotes similar degree of stabilization for the closed hairpin states of both repeat sequences. Temperature-based, smFRET experiments reveal that NaCl favors hairpin closing for (CAG) N/(CAG) N by preordering single-stranded repeat domains to accelerate the closing transition. In contrast, NaCl slows the opening transition of CTG hairpins; however, it promotes misfolded conformations that require unfolding. Energy diagrams illustrate the distinct folding pathways of (CTG) N and (CAG) N repeat domains and identify features that may contribute to their gene-destabilizing effects.
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Affiliation(s)
- Marisa L Mitchell
- Department of Physics , Loyola University Chicago , Chicago , Illinois 60660 , United States
| | - Michael P Leveille
- Department of Physics , Loyola University Chicago , Chicago , Illinois 60660 , United States
| | - Roman S Solecki
- Department of Physics , Loyola University Chicago , Chicago , Illinois 60660 , United States
| | - Thao Tran
- Department of Physics , Loyola University Chicago , Chicago , Illinois 60660 , United States
| | - Brian Cannon
- Department of Physics , Loyola University Chicago , Chicago , Illinois 60660 , United States
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32
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Pešović J, Perić S, Brkušanin M, Brajušković G, Rakočević-Stojanović V, Savić-Pavićević D. Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells. Front Genet 2018; 9:601. [PMID: 30546383 PMCID: PMC6278776 DOI: 10.3389/fgene.2018.00601] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Accepted: 11/15/2018] [Indexed: 12/19/2022] Open
Abstract
CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient's age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells.
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Affiliation(s)
- Jovan Pešović
- Center for Human Molecular Genetics, Faculty of Biology, University of Belgrade, Belgrade, Serbia
| | - Stojan Perić
- School of Medicine, University of Belgrade, Belgrade, Serbia.,Neurology Clinic, Clinical Center of Serbia, Belgrade, Serbia
| | - Miloš Brkušanin
- Center for Human Molecular Genetics, Faculty of Biology, University of Belgrade, Belgrade, Serbia
| | - Goran Brajušković
- Center for Human Molecular Genetics, Faculty of Biology, University of Belgrade, Belgrade, Serbia
| | - Vidosava Rakočević-Stojanović
- School of Medicine, University of Belgrade, Belgrade, Serbia.,Neurology Clinic, Clinical Center of Serbia, Belgrade, Serbia
| | - Dušanka Savić-Pavićević
- Center for Human Molecular Genetics, Faculty of Biology, University of Belgrade, Belgrade, Serbia
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33
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The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA. Genetics 2018; 210:1239-1252. [PMID: 30396881 DOI: 10.1534/genetics.118.301672] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2018] [Accepted: 10/15/2018] [Indexed: 12/13/2022] Open
Abstract
Pathological mutations involving noncoding microsatellite repeats are typically located near promoters in CpG islands and are coupled with extensive repeat instability when sufficiently long. What causes these regions to be prone to repeat instability is not fully understood. There is a general consensus that instability results from the induction of unusual structures in the DNA by the repeats as a consequence of mispairing between complementary strands. In addition, there is some evidence that repeat instability is mediated by RNA transcription through the formation of three-stranded nucleic structures composed of persistent DNA:RNA hybrids, concomitant with single-strand DNA displacements (R-loops). Using human embryonic stem cells with wild-type and repeat expanded alleles in the FMR1 (CGGs) and C9orf72 (GGGGCCs) genes, we show that these loci constitute preferential sites (hotspots) for DNA unpairing. When R-loops are formed, DNA unpairing is more extensive, and is coupled with the interruptions of double-strand structures by the nontranscribing (G-rich) DNA strand. These interruptions are likely to reflect unusual structures in the DNA that drive repeat instability when the G-rich repeats considerably expand. Further, we demonstrate that when the CGGs in FMR1 are hyper-methylated and transcriptionally inactive, local DNA unpairing is abolished. Our study thus takes one more step toward the identification of dynamic, unconventional DNA structures across the G-rich repeats at FMR1 and C9orf72 disease-associated loci.
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34
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Shibata T, Nakatani K. Bicyclic and tricyclic C–C mismatch-binding ligands bind to CCG trinucleotide repeat DNAs. Chem Commun (Camb) 2018; 54:7074-7077. [DOI: 10.1039/c8cc02393j] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
A structural change-inducible ligand that binds to CCG trinucleotide repeats was developed via bivalent interaction and enlarging the aromatic ring system.
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Affiliation(s)
- Tomonori Shibata
- The Institute of Scientific and Industrial Research
- Osaka University
- Ibaraki 567-0047
- Japan
| | - Kazuhiko Nakatani
- The Institute of Scientific and Industrial Research
- Osaka University
- Ibaraki 567-0047
- Japan
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35
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Abstract
The instability of microsatellite DNA repeats is responsible for at least 40 neurodegenerative diseases. Recently, Mirkin and co-workers presented a novel mechanism for microsatellite expansions based on break-induced replication (BIR) at sites of microsatellite-induced replication stalling and fork collapse. The BIR model aims to explain single-step, large expansions of CAG/CTG trinucleotide repeats in dividing cells. BIR has been characterized extensively in Saccharomyces cerevisiae as a mechanism to repair broken DNA replication forks (single-ended DSBs) and degraded telomeric DNA. However, the structural footprints of BIR-like DSB repair have been recognized in human genomic instability and tied to the etiology of diverse developmental diseases; thus, the implications of the paper by Kim et al. (Kim JC, Harris ST, Dinter T, Shah KA, et al., Nat Struct Mol Biol 24: 55-60) extend beyond trinucleotide repeat expansion in yeast and microsatellite instability in human neurological disorders. Significantly, insight into BIR-like repair can explain certain pathways of complex genome rearrangements (CGRs) initiated at non-B form microsatellite DNA in human cancers.
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Affiliation(s)
- Michael Leffak
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH, USA
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36
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Gomes-Pereira M, Monckton DG. Ethidium Bromide Modifies The Agarose Electrophoretic Mobility of CAG•CTG Alternative DNA Structures Generated by PCR. Front Cell Neurosci 2017; 11:153. [PMID: 28611596 PMCID: PMC5447772 DOI: 10.3389/fncel.2017.00153] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2017] [Accepted: 05/10/2017] [Indexed: 12/21/2022] Open
Abstract
The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.
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Affiliation(s)
- Mário Gomes-Pereira
- Laboratory CTGDM, INSERM UMR1163Paris, France.,Institut Imagine, Université Paris Descartes-Sorbonne Paris CitéParis, France
| | - Darren G Monckton
- Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of GlasgowGlasgow, United Kingdom
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van Agtmaal EL, André LM, Willemse M, Cumming SA, van Kessel IDG, van den Broek WJAA, Gourdon G, Furling D, Mouly V, Monckton DG, Wansink DG, Wieringa B. CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing. Mol Ther 2017; 25:24-43. [PMID: 28129118 PMCID: PMC5363205 DOI: 10.1016/j.ymthe.2016.10.014] [Citation(s) in RCA: 95] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2016] [Revised: 10/10/2016] [Accepted: 10/11/2016] [Indexed: 12/15/2022] Open
Abstract
Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.
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Affiliation(s)
- Ellen L van Agtmaal
- Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands
| | - Laurène M André
- Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands
| | - Marieke Willemse
- Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands
| | - Sarah A Cumming
- Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
| | - Ingeborg D G van Kessel
- Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands
| | - Walther J A A van den Broek
- Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands
| | - Geneviève Gourdon
- Inserm UMR 1163, 75015 Paris, France; Imagine Institute, Paris Descartes-Sorbonne Paris Cité University, 75270 Paris, France
| | - Denis Furling
- UPMC Université Paris 06, Inserm UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, 75252 Paris, France
| | - Vincent Mouly
- UPMC Université Paris 06, Inserm UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, 75252 Paris, France
| | - Darren G Monckton
- Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
| | - Derick G Wansink
- Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
| | - Bé Wieringa
- Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
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38
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Jones L, Houlden H, Tabrizi SJ. DNA repair in the trinucleotide repeat disorders. Lancet Neurol 2017; 16:88-96. [PMID: 27979358 DOI: 10.1016/s1474-4422(16)30350-7] [Citation(s) in RCA: 65] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2016] [Revised: 09/22/2016] [Accepted: 10/27/2016] [Indexed: 02/07/2023]
Abstract
BACKGROUND Inherited diseases caused by unstable repeated DNA sequences are rare, but together represent a substantial cause of morbidity. Trinucleotide repeat disorders are severe, usually life-shortening, neurological disorders caused by nucleotide expansions, and most have no disease-modifying treatments. Longer repeat expansions are associated with genetic anticipation (ie, earlier disease onset in successive generations), although the differences in age at onset are not entirely accounted for by repeat length. Such phenotypic variation within disorders implies the existence of additional modifying factors in pathways that can potentially be modulated to treat disease. RECENT DEVELOPMENTS A genome-wide association study detected genetic modifiers of age at onset in Huntington's disease. Similar findings were seen in the spinocerebellar ataxias, indicating an association between DNA damage-response and repair pathways and the age at onset of disease. These studies also suggest that a common genetic mechanism modulates age at onset across polyglutamine diseases and could extend to other repeat expansion disorders. Genetic defects in DNA repair underlie other neurodegenerative disorders (eg, ataxia-telangiectasia), and DNA double-strand breaks are crucial to the modulation of early gene expression, which provides a mechanistic link between DNA repair and neurodegeneration. Mismatch and base-excision repair are important in the somatic expansion of repeated sequences in mouse models of trinucleotide repeat disorders, and somatic expansion of the expanded CAG tract in HTT correlates with age at onset of Huntington's disease and other trinucleotide repeat disorders. WHERE NEXT?: To understand the common genetic architecture of trinucleotide repeat disorders and any further genetic susceptibilities in individual disorders, genetic analysis with increased numbers of variants and sample sizes is needed, followed by sequencing approaches to define the phenotype-modifying variants. The findings must then be translated into cell biology analyses to elucidate the mechanisms through which the genetic variants operate. Genes that have roles in the DNA damage response could underpin a common DNA repeat-based mechanism and provide new therapeutic targets (and hence therapeutics) in multiple trinucleotide repeat disorders.
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Affiliation(s)
- Lesley Jones
- MRC Centre for Neuropsychiatric Genetics and Genomics, Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, UK.
| | - Henry Houlden
- Department of Molecular Neuroscience and MRC Centre for Neuromuscular Diseases, Institute of Neurology, Queen Square, London, UK
| | - Sarah J Tabrizi
- UCL Huntington's Disease Centre, Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, UK
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39
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Gadgil R, Barthelemy J, Lewis T, Leffak M. Replication stalling and DNA microsatellite instability. Biophys Chem 2016; 225:38-48. [PMID: 27914716 DOI: 10.1016/j.bpc.2016.11.007] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Revised: 11/05/2016] [Accepted: 11/05/2016] [Indexed: 01/08/2023]
Abstract
Microsatellites are short, tandemly repeated DNA motifs of 1-6 nucleotides, also termed simple sequence repeats (SRSs) or short tandem repeats (STRs). Collectively, these repeats comprise approximately 3% of the human genome Subramanian et al. (2003), Lander and Lander (2001) [1,2], and represent a large reservoir of loci highly prone to mutations Sun et al. (2012), Ellegren (2004) [3,4] that contribute to human evolution and disease. Microsatellites are known to stall and reverse replication forks in model systems Pelletier et al. (2003), Samadashwily et al. (1997), Kerrest et al. (2009) [5-7], and are hotspots of chromosomal double strand breaks (DSBs). We briefly review the relationship of these repeated sequences to replication stalling and genome instability, and present recent data on the impact of replication stress on DNA fragility at microsatellites in vivo.
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Affiliation(s)
- R Gadgil
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA
| | - J Barthelemy
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA
| | - T Lewis
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA
| | - M Leffak
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
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40
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Cinesi C, Aeschbach L, Yang B, Dion V. Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase. Nat Commun 2016; 7:13272. [PMID: 27827362 PMCID: PMC5105158 DOI: 10.1038/ncomms13272] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2016] [Accepted: 09/12/2016] [Indexed: 12/15/2022] Open
Abstract
CAG/CTG repeat expansions cause over 13 neurological diseases that remain without a cure. Because longer tracts cause more severe phenotypes, contracting them may provide a therapeutic avenue. No currently known agent can specifically generate contractions. Using a GFP-based chromosomal reporter that monitors expansions and contractions in the same cell population, here we find that inducing double-strand breaks within the repeat tract causes instability in both directions. In contrast, the CRISPR-Cas9 D10A nickase induces mainly contractions independently of single-strand break repair. Nickase-induced contractions depend on the DNA damage response kinase ATM, whereas ATR inhibition increases both expansions and contractions in a MSH2- and XPA-dependent manner. We propose that DNA gaps lead to contractions and that the type of DNA damage present within the repeat tract dictates the levels and the direction of CAG repeat instability. Our study paves the way towards deliberate induction of CAG/CTG repeat contractions in vivo.
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Affiliation(s)
- Cinzia Cinesi
- Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland
| | - Lorène Aeschbach
- Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland
| | - Bin Yang
- Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland
| | - Vincent Dion
- Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland
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41
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Viterbo D, Michoud G, Mosbach V, Dujon B, Richard GF. Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair. DNA Repair (Amst) 2016; 42:94-106. [DOI: 10.1016/j.dnarep.2016.03.002] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Revised: 02/01/2016] [Accepted: 03/11/2016] [Indexed: 10/22/2022]
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42
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Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks. DNA Repair (Amst) 2016; 42:107-18. [PMID: 27155933 DOI: 10.1016/j.dnarep.2016.04.002] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 03/22/2016] [Accepted: 04/05/2016] [Indexed: 11/22/2022]
Abstract
Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats.
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43
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Schmidt MHM, Pearson CE. Disease-associated repeat instability and mismatch repair. DNA Repair (Amst) 2015; 38:117-126. [PMID: 26774442 DOI: 10.1016/j.dnarep.2015.11.008] [Citation(s) in RCA: 160] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2015] [Revised: 11/23/2015] [Accepted: 11/30/2015] [Indexed: 12/15/2022]
Abstract
Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.
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Affiliation(s)
- Monika H M Schmidt
- Genetics & Genome Biology, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay St., Toronto, Ontario M5G 0A4, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada
| | - Christopher E Pearson
- Genetics & Genome Biology, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay St., Toronto, Ontario M5G 0A4, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
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44
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Zamiri B, Mirceta M, Bomsztyk K, Macgregor RB, Pearson CE. Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8•(GGCCCC)8 repeat: effect of CpG methylation. Nucleic Acids Res 2015; 43:10055-64. [PMID: 26432832 PMCID: PMC4787773 DOI: 10.1093/nar/gkv1008] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Accepted: 09/22/2015] [Indexed: 12/13/2022] Open
Abstract
Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG methylated with unknown consequences. Typically, quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and electrophoresis, we characterized the structures formed by (GGGGCC)8 and (GGCCCC)8 strands with and without 5-methylcytosine (5mCpG) or 5-hydroxymethylcytosine (5hmCpG) methylation. All strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in K(+), Na(+) or Li(+)), but no feature typical of i-motifs. C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position, hence C9orf72 quadruplexes still formed when N7-deazaGuanine replace all Guanines. 5mCpG and 5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift analysis, bound C-rich but not G-rich strands, with a binding preference of unmethylated > 5hmCpG > 5mCpG, where methylated DNA-protein complexes were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich sequences interspersed with G-residues, one must consider quadruplex formation and that methylation of quadruplexes may affect epigenetic processes.
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Affiliation(s)
- Bita Zamiri
- Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada
| | - Mila Mirceta
- Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada
| | - Karol Bomsztyk
- UW Medicine South Lake Union, University of Washington, Seattle WA 98109, USA
| | - Robert B Macgregor
- Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada
| | - Christopher E Pearson
- Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1, Canada
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45
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Yanovsky-Dagan S, Mor-Shaked H, Eiges R. Modeling diseases of noncoding unstable repeat expansions using mutant pluripotent stem cells. World J Stem Cells 2015; 7:823-838. [PMID: 26131313 PMCID: PMC4478629 DOI: 10.4252/wjsc.v7.i5.823] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2014] [Revised: 02/22/2015] [Accepted: 04/07/2015] [Indexed: 02/06/2023] Open
Abstract
Pathogenic mutations involving DNA repeat expansions are responsible for over 20 different neuronal and neuromuscular diseases. All result from expanded tracts of repetitive DNA sequences (mostly microsatellites) that become unstable beyond a critical length when transmitted across generations. Nearly all are inherited as autosomal dominant conditions and are typically associated with anticipation. Pathologic unstable repeat expansions can be classified according to their length, repeat sequence, gene location and underlying pathologic mechanisms. This review summarizes the current contribution of mutant pluripotent stem cells (diseased human embryonic stem cells and patient-derived induced pluripotent stem cells) to the research of unstable repeat pathologies by focusing on particularly large unstable noncoding expansions. Among this class of disorders are Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome, myotonic dystrophy type 1 and myotonic dystrophy type 2, Friedreich ataxia and C9 related amyotrophic lateral sclerosis and/or frontotemporal dementia, Facioscapulohumeral Muscular Dystrophy and potentially more. Common features that are typical to this subclass of conditions are RNA toxic gain-of-function, epigenetic loss-of-function, toxic repeat-associated non-ATG translation and somatic instability. For each mechanism we summarize the currently available stem cell based models, highlight how they contributed to better understanding of the related mechanism, and discuss how they may be utilized in future investigations.
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Sun S, Zhang W, Chen X, Song H. The CAA repeat polymorphism in the ZFHX3 gene is associated with risk of coronary heart disease in a Chinese population. TOHOKU J EXP MED 2015; 235:261-6. [PMID: 25797214 DOI: 10.1620/tjem.235.261] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Coronary heart disease (CHD) is a disease resulting from the interaction between genetic variations and environmental factors. Zinc finger homeobox 3 (ZFHX3) is a transcription factor and contains a poly-glutamine tract in a compositionally biased region that is encoded by exon 9, containing a cluster of CAG and CAA triplets followed by the polymorphic CAA repeats: (CAG)2(CAA)2(CAG)3CAACAG(CAA)nGCA. Thus, nine successive glutamine residues precede the poly-glutamine tract, encoded by the polymorphic CAA repeats. The aim of this study was to investigate the association of the CAA repeat polymorphism in exon 9 of the ZFHX3 gene with the risk of CHD in a Chinese population. The CAA repeat polymorphism was determined by polymerase chain reaction followed by DNA sequencing in 321 CHD patients. Genotype frequencies were compared using the non-parametric mood median test. Four alleles of CAG(CAA)10GCA, CAG(CAA)8GCA, CAG(CAA)9GCA, and CAG(CAA)11GCA were found in Chinese CHD patients in exon 9 of the ZFHX3 gene. The CAG(CAA)10GCA was a major allele (95.95%), and the CAG(CAA)8GCA was a minor allele (3.58%). The CAG(CAA)9GCA and CAG(CAA)11GCA were rare alleles (0.31% and 0.16%). The CAG(CAA)10GCA allele encodes a poly-glutamine tract of 19 residues. Importantly, the CHD patients homozygous for the CAG(CAA)10GCA allele had a higher risk of CHD, compared to the heterozygous patients carrying a CAG(CAA)8GCA allele. Moreover, the CAG(CAA)10GCA allele was significantly associated with hypertension, diabetes mellitus, or dyslipidemia (P < 0.05). Thus, the CAA repeat polymorphism in exon 9 of the ZFHX3 gene contributes to the CHD susceptibility in the Chinese population.
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Affiliation(s)
- Shunchang Sun
- Central Laboratory, Shenzhen Baoan Hospital, Southern Medical University
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Richard GF. Shortening trinucleotide repeats using highly specific endonucleases: a possible approach to gene therapy? Trends Genet 2015; 31:177-86. [PMID: 25743488 DOI: 10.1016/j.tig.2015.02.003] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2014] [Revised: 02/04/2015] [Accepted: 02/05/2015] [Indexed: 12/31/2022]
Abstract
Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.
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Affiliation(s)
- Guy-Franck Richard
- Institut Pasteur, Department Genomes and Genetics, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 3525, 25 Rue du Dr Roux, 75015 Paris, France
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Abstract
DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSβ and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway.
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Affiliation(s)
- Ravi R Iyer
- Teva Branded Pharmaceutical Products R&D, Inc., West Chester, Pennsylvania 19380;
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Reddy K, Schmidt MHM, Geist JM, Thakkar NP, Panigrahi GB, Wang YH, Pearson CE. Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability. Nucleic Acids Res 2014; 42:10473-87. [PMID: 25147206 PMCID: PMC4176329 DOI: 10.1093/nar/gku658] [Citation(s) in RCA: 78] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.
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Affiliation(s)
- Kaalak Reddy
- Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada
| | - Monika H M Schmidt
- Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada
| | - Jaimie M Geist
- Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Department of Biology, Laurentian University, Sudbury, Ontario P3E 2C6, Canada
| | - Neha P Thakkar
- Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada
| | - Gagan B Panigrahi
- Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada
| | - Yuh-Hwa Wang
- Department of Biochemistry & Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
| | - Christopher E Pearson
- Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada
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Modifiers of (CAG)n instability in Machado–Joseph disease (MJD/SCA3) transmissions: an association study with DNA replication, repair and recombination genes. Hum Genet 2014; 133:1311-8. [DOI: 10.1007/s00439-014-1467-8] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2014] [Accepted: 07/03/2014] [Indexed: 12/24/2022]
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