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1
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Casazza KM, Williams GM, Johengen L, Twoey G, Surtees JA. Msh2-Msh3 DNA-binding is not sufficient to promote trinucleotide repeat expansions in Saccharomyces cerevisiae. Genetics 2025; 229:iyae222. [PMID: 39790027 PMCID: PMC11912836 DOI: 10.1093/genetics/iyae222] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Accepted: 12/25/2024] [Indexed: 01/12/2025] Open
Abstract
Mismatch repair (MMR) is a highly conserved DNA repair pathway that recognizes mispairs that occur spontaneously during DNA replication and coordinates their repair. In Saccharomyces cerevisiae, Msh2-Msh3 and Msh2-Msh6 initiate MMR by recognizing and binding insertion or deletion (in/del) loops up to ∼17 nucleotides (nt.) and base-base mispairs, respectively; the 2 complexes have overlapping specificity for small (1-2 nt.) in/dels. The DNA-binding specificity for the 2 complexes resides in their respective mispair binding domains (MBDs) and has distinct DNA-binding modes. Msh2-Msh3 also plays a role in promoting CAG/CTG trinucleotide repeat (TNR) expansions, which underlie many neurodegenerative diseases such as Huntington's disease and myotonic dystrophy type 1. Models for Msh2-Msh3's role in promoting TNR tract expansion have invoked its specific DNA-binding activity and predict that the TNR structure alters its DNA binding and downstream activities to block repair. Using a chimeric Msh complex that replaces the MBD of Msh6 with the Msh3 MBD, we demonstrate that Msh2-Msh3 DNA-binding activity is not sufficient to promote TNR expansions. We propose a model for Msh2-Msh3-mediated TNR expansions that requires a fully functional Msh2-Msh3 including DNA binding, coordinated ATP binding, and hydrolysis activities and interactions with Mlh complexes that are analogous to those required for MMR.
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Affiliation(s)
- Katherine M Casazza
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
| | - Gregory M Williams
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
- Curia Global, Inc., Buffalo, NY 14203, USA
| | - Lauren Johengen
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
| | - Gavin Twoey
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
| | - Jennifer A Surtees
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
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2
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Pan F, Xu P, Roland C, Sagui C, Weninger K. Structural and Dynamical Properties of Nucleic Acid Hairpins Implicated in Trinucleotide Repeat Expansion Diseases. Biomolecules 2024; 14:1278. [PMID: 39456210 PMCID: PMC11505666 DOI: 10.3390/biom14101278] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2024] [Revised: 09/26/2024] [Accepted: 10/05/2024] [Indexed: 10/28/2024] Open
Abstract
Dynamic mutations in some human genes containing trinucleotide repeats are associated with severe neurodegenerative and neuromuscular disorders-known as Trinucleotide (or Triplet) Repeat Expansion Diseases (TREDs)-which arise when the repeat number of triplets expands beyond a critical threshold. While the mechanisms causing the DNA triplet expansion are complex and remain largely unknown, it is now recognized that the expandable repeats lead to the formation of nucleotide configurations with atypical structural characteristics that play a crucial role in TREDs. These nonstandard nucleic acid forms include single-stranded hairpins, Z-DNA, triplex structures, G-quartets and slipped-stranded duplexes. Of these, hairpin structures are the most prolific and are associated with the largest number of TREDs and have therefore been the focus of recent single-molecule FRET experiments and molecular dynamics investigations. Here, we review the structural and dynamical properties of nucleic acid hairpins that have emerged from these studies and the implications for repeat expansion mechanisms. The focus will be on CAG, GAC, CTG and GTC hairpins and their stems, their atomistic structures, their stability, and the important role played by structural interrupts.
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Affiliation(s)
- Feng Pan
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
- Department of Statistics, Florida State University, Tallahassee, FL 32306, USA
| | - Pengning Xu
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Christopher Roland
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
| | - Celeste Sagui
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
| | - Keith Weninger
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
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3
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Mätlik K, Baffuto M, Kus L, Deshmukh AL, Davis DA, Paul MR, Carroll TS, Caron MC, Masson JY, Pearson CE, Heintz N. Cell-type-specific CAG repeat expansions and toxicity of mutant Huntingtin in human striatum and cerebellum. Nat Genet 2024; 56:383-394. [PMID: 38291334 PMCID: PMC10937393 DOI: 10.1038/s41588-024-01653-6] [Citation(s) in RCA: 42] [Impact Index Per Article: 42.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2023] [Accepted: 12/28/2023] [Indexed: 02/01/2024]
Abstract
Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships among CAG expansions, death of specific cell types and molecular events associated with these processes are not established. Here, we used fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise at mHTT in striatal medium spiny neurons (MSNs), cholinergic interneurons and cerebellar Purkinje neurons, and at mutant ATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSβ), which can inhibit nucleolytic excision of CAG slip-outs by FAN1. Our data support a model in which CAG expansions are necessary but may not be sufficient for cell death and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.
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Affiliation(s)
- Kert Mätlik
- Laboratory of Molecular Biology, The Rockefeller University, New York, NY, USA
| | - Matthew Baffuto
- Laboratory of Molecular Biology, The Rockefeller University, New York, NY, USA
| | - Laura Kus
- Laboratory of Molecular Biology, The Rockefeller University, New York, NY, USA
| | - Amit Laxmikant Deshmukh
- Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
| | - David A Davis
- Miller School of Medicine, University of Miami, Miami, FL, USA
| | - Matthew R Paul
- Bioinformatics Resource Center, The Rockefeller University, New York, NY, USA
| | - Thomas S Carroll
- Bioinformatics Resource Center, The Rockefeller University, New York, NY, USA
| | - Marie-Christine Caron
- CHU de Québec Research Center, Oncology Division, Laval University Cancer Research Center, Quebec City, Quebec, Canada
| | - Jean-Yves Masson
- CHU de Québec Research Center, Oncology Division, Laval University Cancer Research Center, Quebec City, Quebec, Canada
| | - Christopher E Pearson
- Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
- Program of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Nathaniel Heintz
- Laboratory of Molecular Biology, The Rockefeller University, New York, NY, USA.
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4
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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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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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Singh AK, Amar I, Ramadasan H, Kappagantula KS, Chavali S. Proteins with amino acid repeats constitute a rapidly evolvable and human-specific essentialome. Cell Rep 2023; 42:112811. [PMID: 37453061 DOI: 10.1016/j.celrep.2023.112811] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Revised: 05/30/2023] [Accepted: 06/29/2023] [Indexed: 07/18/2023] Open
Abstract
Protein products of essential genes, indispensable for organismal survival, are highly conserved and bring about fundamental functions. Interestingly, proteins that contain amino acid homorepeats that tend to evolve rapidly are enriched in eukaryotic essentialomes. Why are proteins with hypermutable homorepeats enriched in conserved and functionally vital essential proteins? We solve this functional versus evolutionary paradox by demonstrating that human essential proteins with homorepeats bring about crosstalk across biological processes through high interactability and have distinct regulatory functions affecting expansive global regulation. Importantly, essential proteins with homorepeats rapidly diverge with the amino acid substitutions frequently affecting functional sites, likely facilitating rapid adaptability. Strikingly, essential proteins with homorepeats influence human-specific embryonic and brain development, implying that the presence of homorepeats could contribute to the emergence of human-specific processes. Thus, we propose that homorepeat-containing essential proteins affecting species-specific traits can be potential intervention targets across pathologies, including cancers and neurological disorders.
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Affiliation(s)
- Anjali K Singh
- Department of Biology, Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati 517507, Andhra Pradesh, India
| | - Ishita Amar
- Department of Biology, Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati 517507, Andhra Pradesh, India
| | - Harikrishnan Ramadasan
- Department of Biology, Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati 517507, Andhra Pradesh, India
| | - Keertana S Kappagantula
- Department of Biology, Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati 517507, Andhra Pradesh, India
| | - Sreenivas Chavali
- Department of Biology, Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati 517507, Andhra Pradesh, India.
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7
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Mätlik K, Baffuto M, Kus L, Deshmukh AL, Davis DA, Paul MR, Carroll TS, Caron MC, Masson JY, Pearson CE, Heintz N. Cell Type Specific CAG Repeat Expansions and Toxicity of Mutant Huntingtin in Human Striatum and Cerebellum. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.24.538082. [PMID: 37333326 PMCID: PMC10274669 DOI: 10.1101/2023.04.24.538082] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/20/2023]
Abstract
Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSβ), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.
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8
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Abstract
Repetitive elements in the human genome, once considered 'junk DNA', are now known to adopt more than a dozen alternative (that is, non-B) DNA structures, such as self-annealed hairpins, left-handed Z-DNA, three-stranded triplexes (H-DNA) or four-stranded guanine quadruplex structures (G4 DNA). These dynamic conformations can act as functional genomic elements involved in DNA replication and transcription, chromatin organization and genome stability. In addition, recent studies have revealed a role for these alternative structures in triggering error-generating DNA repair processes, thereby actively enabling genome plasticity. As a driving force for genetic variation, non-B DNA structures thus contribute to both disease aetiology and evolution.
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Affiliation(s)
- Guliang Wang
- Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Dell Paediatric Research Institute, Austin, TX, USA
| | - Karen M Vasquez
- Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Dell Paediatric Research Institute, Austin, TX, USA.
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9
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Masnovo C, Lobo AF, Mirkin SM. Replication dependent and independent mechanisms of GAA repeat instability. DNA Repair (Amst) 2022; 118:103385. [PMID: 35952488 PMCID: PMC9675320 DOI: 10.1016/j.dnarep.2022.103385] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 07/28/2022] [Accepted: 07/30/2022] [Indexed: 11/20/2022]
Abstract
Trinucleotide repeat instability is a driver of human disease. Large expansions of (GAA)n repeats in the first intron of the FXN gene are the cause Friedreich's ataxia (FRDA), a progressive degenerative disorder which cannot yet be prevented or treated. (GAA)n repeat instability arises during both replication-dependent processes, such as cell division and intergenerational transmission, as well as in terminally differentiated somatic tissues. Here, we provide a brief historical overview on the discovery of (GAA)n repeat expansions and their association to FRDA, followed by recent advances in the identification of triplex H-DNA formation and replication fork stalling. The main body of this review focuses on the last decade of progress in understanding the mechanism of (GAA)n repeat instability during DNA replication and/or DNA repair. We propose that the discovery of additional mechanisms of (GAA)n repeat instability can be achieved via both comparative approaches to other repeat expansion diseases and genome-wide association studies. Finally, we discuss the advances towards FRDA prevention or amelioration that specifically target (GAA)n repeat expansions.
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Affiliation(s)
- Chiara Masnovo
- Department of Biology, Tufts University, Medford, MA 02155, USA
| | - Ayesha F Lobo
- Department of Biology, Tufts University, Medford, MA 02155, USA
| | - Sergei M Mirkin
- Department of Biology, Tufts University, Medford, MA 02155, USA.
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10
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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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11
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12
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Chan KY, Li X, Ortega J, Gu L, Li GM. DNA polymerase θ promotes CAG•CTG repeat expansions in Huntington's disease via insertion sequences of its catalytic domain. J Biol Chem 2021; 297:101144. [PMID: 34473992 PMCID: PMC8463855 DOI: 10.1016/j.jbc.2021.101144] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Revised: 08/23/2021] [Accepted: 08/27/2021] [Indexed: 12/04/2022] Open
Abstract
Huntington's disease (HD), a neurodegenerative disease characterized by progressive dementia, psychiatric problems, and chorea, is known to be caused by CAG repeat expansions in the HD gene HTT. However, the mechanism of this pathology is not fully understood. The translesion DNA polymerase θ (Polθ) carries a large insertion sequence in its catalytic domain, which has been shown to allow DNA loop-outs in the primer strand. As a result of high levels of oxidative DNA damage in neural cells and Polθ's subsequent involvement in base excision repair of oxidative DNA damage, we hypothesized that Polθ contributes to CAG repeat expansion while repairing oxidative damage within HTT. Here, we performed Polθ-catalyzed in vitro DNA synthesis using various CAG•CTG repeat DNA substrates that are similar to base excision repair intermediates. We show that Polθ efficiently extends (CAG)n•(CTG)n hairpin primers, resulting in hairpin retention and repeat expansion. Polθ also triggers repeat expansions to pass the threshold for HD when the DNA template contains 35 repeats upward. Strikingly, Polθ depleted of the catalytic insertion fails to induce repeat expansions regardless of primers and templates used, indicating that the insertion sequence is responsible for Polθ's error-causing activity. In addition, the level of chromatin-bound Polθ in HD cells is significantly higher than in non-HD cells and exactly correlates with the degree of CAG repeat expansion, implying Polθ's involvement in triplet repeat instability. Therefore, we have identified Polθ as a potent factor that promotes CAG•CTG repeat expansions in HD and other neurodegenerative disorders.
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Affiliation(s)
- Kara Y Chan
- Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA; Department of Toxicology and Cancer Biology, University of Kentucky College of Medicine, Lexington, Kentucky, USA
| | - Xueying Li
- Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Janice Ortega
- Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Liya Gu
- Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Guo-Min Li
- Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA; Department of Toxicology and Cancer Biology, University of Kentucky College of Medicine, Lexington, Kentucky, USA.
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13
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Chatain J, Blond A, Phan AT, Saintomé C, Alberti P. GGGCTA repeats can fold into hairpins poorly unfolded by replication protein A: a possible origin of the length-dependent instability of GGGCTA variant repeats in human telomeres. Nucleic Acids Res 2021; 49:7588-7601. [PMID: 34214172 PMCID: PMC8287962 DOI: 10.1093/nar/gkab518] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2020] [Revised: 06/01/2021] [Accepted: 06/30/2021] [Indexed: 11/19/2022] Open
Abstract
Human telomeres are composed of GGGTTA repeats and interspersed with variant repeats. The GGGCTA variant motif was identified in the proximal regions of human telomeres about 10 years ago and was shown to display a length-dependent instability. In parallel, a structural study showed that four GGGCTA repeats folded into a non-canonical G-quadruplex (G4) comprising a Watson-Crick GCGC tetrad. It was proposed that this non-canonical G4 might be an additional obstacle for telomere replication. In the present study, we demonstrate that longer GGGCTA arrays fold into G4 and into hairpins. We also demonstrate that replication protein A (RPA) efficiently binds to GGGCTA repeats structured into G4 but poorly binds to GGGCTA repeats structured into hairpins. Our results (along with results obtained with a more stable variant motif) suggest that GGGCTA hairpins are at the origin of GGGCTA length-dependent instability. They also suggest, as working hypothesis, that failure of efficient binding of RPA to GGGCTA structured into hairpins might be involved in the mechanism of GGGCTA array instability. On the basis of our present and past studies about telomeric G4 and their interaction with RPA, we propose an original point of view about telomeric G4 and the evolution of telomeric motifs.
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Affiliation(s)
- Jean Chatain
- Laboratoire Structure et Instabilité des Génomes (StrInG), Muséum national d’Histoire naturelle, CNRS, Inserm, Paris 75005, France
| | - Alain Blond
- Laboratoire Molécules de Communication et Adaptation des Microorganismes (MCAM), Muséum national d’Histoire naturelle, CNRS, Paris 75005, France
| | - Anh Tuân Phan
- School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371, Singapore
- NTU Institute of Structural Biology, Nanyang Technological University, Singapore 636921, Singapore
| | - Carole Saintomé
- Laboratoire Structure et Instabilité des Génomes (StrInG), Muséum national d’Histoire naturelle, CNRS, Inserm, Paris 75005, France
- Sorbonne Université, UFR927, Paris 75005, France
| | - Patrizia Alberti
- Laboratoire Structure et Instabilité des Génomes (StrInG), Muséum national d’Histoire naturelle, CNRS, Inserm, Paris 75005, France
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14
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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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15
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Replication-independent instability of Friedreich's ataxia GAA repeats during chronological aging. Proc Natl Acad Sci U S A 2021; 118:2013080118. [PMID: 33495349 PMCID: PMC7865128 DOI: 10.1073/pnas.2013080118] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The inheritance of long (GAA)n repeats in the frataxin gene causes the debilitating neurodegenerative disease Friedreich’s ataxia. Subsequent expansions of these repeats throughout a patient’s lifetime in the affected tissues, like the nervous system, may contribute to disease onset. We developed an experimental model to characterize the mechanisms of repeat instability in nondividing cells to better understand how mutations can occur as cells age chronologically. We show that repeats can expand in nondividing cells. Notably, however, large deletions are the major type of repeat-mediated genome instability in nondividing cells, implicating the loss of important genetic material with aging in the progression of Friedreich’s ataxia. Nearly 50 hereditary diseases result from the inheritance of abnormally long repetitive DNA microsatellites. While it was originally believed that the size of inherited repeats is the key factor in disease development, it has become clear that somatic instability of these repeats throughout an individual’s lifetime strongly contributes to disease onset and progression. Importantly, somatic instability is commonly observed in terminally differentiated, postmitotic cells, such as neurons. To unravel the mechanisms of repeat instability in nondividing cells, we created an experimental system to analyze the mutability of Friedreich’s ataxia (GAA)n repeats during chronological aging of quiescent Saccharomyces cerevisiae. Unexpectedly, we found that the predominant repeat-mediated mutation in nondividing cells is large-scale deletions encompassing parts, or the entirety, of the repeat and adjacent regions. These deletions are caused by breakage at the repeat mediated by mismatch repair (MMR) complexes MutSβ and MutLα and DNA endonuclease Rad1, followed by end-resection by Exo1 and repair of the resulting double-strand breaks (DSBs) via nonhomologous end joining. We also observed repeat-mediated gene conversions as a result of DSB repair via ectopic homologous recombination during chronological aging. Repeat expansions accrue during chronological aging as well—particularly in the absence of MMR-induced DSBs. These expansions depend on the processivity of DNA polymerase δ while being counteracted by Exo1 and MutSβ, implicating nick repair. Altogether, these findings show that the mechanisms and types of (GAA)n repeat instability differ dramatically between dividing and nondividing cells, suggesting that distinct repeat-mediated mutations in terminally differentiated somatic cells might influence Friedreich’s ataxia pathogenesis.
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16
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Richard GF. The Startling Role of Mismatch Repair in Trinucleotide Repeat Expansions. Cells 2021; 10:cells10051019. [PMID: 33925919 PMCID: PMC8145212 DOI: 10.3390/cells10051019] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 04/20/2021] [Accepted: 04/21/2021] [Indexed: 12/26/2022] Open
Abstract
Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.
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Affiliation(s)
- Guy-Franck Richard
- Institut Pasteur, CNRS UMR3525, 25 rue du Docteur Roux, 75015 Paris, France
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17
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Crosstalk between Different DNA Repair Pathways Contributes to Neurodegenerative Diseases. BIOLOGY 2021; 10:biology10020163. [PMID: 33669593 PMCID: PMC7922961 DOI: 10.3390/biology10020163] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/31/2021] [Revised: 02/11/2021] [Accepted: 02/16/2021] [Indexed: 02/07/2023]
Abstract
Simple Summary Constant exposure to endogenous and environmental factors induces oxidative stress and DNA damage. Rare brain disorders caused by defects in DNA repair and DNA damage response (DDR) signaling establish that failure to process DNA damage may lead to neurodegeneration. In this review, we present mechanisms that link DDR with neurodegeneration in these disorders and discuss their relevance for common age-related neurodegenerative diseases (NDDs). Moreover, we highlight recent insight into the crosstalk between the DDR and other cellular processes known to be disturbed during NDDs. Abstract Genomic integrity is maintained by DNA repair and the DNA damage response (DDR). Defects in certain DNA repair genes give rise to many rare progressive neurodegenerative diseases (NDDs), such as ocular motor ataxia, Huntington disease (HD), and spinocerebellar ataxias (SCA). Dysregulation or dysfunction of DDR is also proposed to contribute to more common NDDs, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Here, we present mechanisms that link DDR with neurodegeneration in rare NDDs caused by defects in the DDR and discuss the relevance for more common age-related neurodegenerative diseases. Moreover, we highlight recent insight into the crosstalk between the DDR and other cellular processes known to be disturbed during NDDs. We compare the strengths and limitations of established model systems to model human NDDs, ranging from C. elegans and mouse models towards advanced stem cell-based 3D models.
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18
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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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19
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Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats. Nat Commun 2021; 12:204. [PMID: 33420051 PMCID: PMC7794359 DOI: 10.1038/s41467-020-20426-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Accepted: 11/30/2020] [Indexed: 12/18/2022] Open
Abstract
Expansions of CAG/CTG trinucleotide repeats in DNA are the cause of at least 17 degenerative human disorders, including Huntington’s Disease. Repeat instability is thought to occur via the formation of intrastrand hairpins during replication, repair, recombination, and transcription though relatively little is known about their structure and dynamics. We use single-molecule Förster resonance energy transfer to study DNA three-way junctions (3WJs) containing slip-outs composed of CAG or CTG repeats. 3WJs that only have repeats in the slip-out show two-state behavior, which we attribute to conformational flexibility at the 3WJ branchpoint. When the triplet repeats extend into the adjacent duplex, additional dynamics are observed, which we assign to interconversion of positional isomers. We propose a branchpoint migration model that involves conformational rearrangement, strand exchange, and bulge-loop movement. This migration has implications for how repeat slip-outs are processed by the cellular machinery, disease progression, and their development as drug targets. DNA three-way junctions are branched structures formed during replication, repair, and recombination, and are involved in models of repeat expansion. Here the authors use single-molecule Förster resonance energy transfer to reveal the dynamics of DNA three-way junctions containing slip-outs composed of CAG or CTG repeats.
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20
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HDAC3 deacetylates the DNA mismatch repair factor MutSβ to stimulate triplet repeat expansions. Proc Natl Acad Sci U S A 2020; 117:23597-23605. [PMID: 32900932 PMCID: PMC7519323 DOI: 10.1073/pnas.2013223117] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Trinucleotide repeat (TNR) expansions cause nearly 20 severe human neurological diseases which are currently untreatable. For some of these diseases, ongoing somatic expansions accelerate disease progression and may influence age of onset. This new knowledge emphasizes the importance of understanding the protein factors that drive expansions. Recent genetic evidence indicates that the mismatch repair factor MutSβ (Msh2-Msh3 complex) and the histone deacetylase HDAC3 function in the same pathway to drive triplet repeat expansions. Here we tested the hypothesis that HDAC3 deacetylates MutSβ and thereby activates it to drive expansions. The HDAC3-selective inhibitor RGFP966 was used to examine its biological and biochemical consequences in human tissue culture cells. HDAC3 inhibition efficiently suppresses repeat expansion without impeding canonical mismatch repair activity. Five key lysine residues in Msh3 are direct targets of HDAC3 deacetylation. In cells expressing Msh3 in which these lysine residues are mutated to arginine, the inhibitory effect of RGFP966 on expansions is largely bypassed, consistent with the direct deacetylation hypothesis. RGFP966 treatment does not alter MutSβ subunit abundance or complex formation but does partially control its subcellular localization. Deacetylation sites in Msh3 overlap a nuclear localization signal, and we show that localization of MutSβ is partially dependent on HDAC3 activity. Together, these results indicate that MutSβ is a key target of HDAC3 deacetylation and provide insights into an innovative regulatory mechanism for triplet repeat expansions. The results suggest expansion activity may be druggable and support HDAC3-selective inhibition as an attractive therapy in some triplet repeat expansion diseases.
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21
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Ain Q, Schmeer C, Wengerodt D, Witte OW, Kretz A. Extrachromosomal Circular DNA: Current Knowledge and Implications for CNS Aging and Neurodegeneration. Int J Mol Sci 2020; 21:E2477. [PMID: 32252492 PMCID: PMC7177960 DOI: 10.3390/ijms21072477] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Revised: 03/26/2020] [Accepted: 03/30/2020] [Indexed: 12/13/2022] Open
Abstract
Still unresolved is the question of how a lifetime accumulation of somatic gene copy number alterations impact organ functionality and aging and age-related pathologies. Such an issue appears particularly relevant in the broadly post-mitotic central nervous system (CNS), where non-replicative neurons are restricted in DNA-repair choices and are prone to accumulate DNA damage, as they remain unreplaced over a lifetime. Both DNA injuries and consecutive DNA-repair strategies are processes that can evoke extrachromosomal circular DNA species, apparently from either part of the genome. Due to their capacity to amplify gene copies and related transcripts, the individual cellular load of extrachromosomal circular DNAs will contribute to a dynamic pool of additional coding and regulatory chromatin elements. Analogous to tumor tissues, where the mosaicism of circular DNAs plays a well-characterized role in oncogene plasticity and drug resistance, we suggest involvement of the "circulome" also in the CNS. Accordingly, we summarize current knowledge on the molecular biogenesis, homeostasis and gene regulatory impacts of circular extrachromosomal DNA and propose, in light of recent discoveries, a critical role in CNS aging and neurodegeneration. Future studies will elucidate the influence of individual extrachromosomal DNA species according to their sequence complexity and regional distribution or cell-type-specific abundance.
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Affiliation(s)
- Quratul Ain
- Hans-Berger Department of Neurology, Jena University Hospital, 07747 Jena, Thuringia, Germany; (Q.A.); (C.S.); (D.W.); (O.W.W.)
| | - Christian Schmeer
- Hans-Berger Department of Neurology, Jena University Hospital, 07747 Jena, Thuringia, Germany; (Q.A.); (C.S.); (D.W.); (O.W.W.)
- Jena Center for Healthy Ageing, Jena University Hospital, 07747 Jena, Thuringia, Germany
| | - Diane Wengerodt
- Hans-Berger Department of Neurology, Jena University Hospital, 07747 Jena, Thuringia, Germany; (Q.A.); (C.S.); (D.W.); (O.W.W.)
| | - Otto W. Witte
- Hans-Berger Department of Neurology, Jena University Hospital, 07747 Jena, Thuringia, Germany; (Q.A.); (C.S.); (D.W.); (O.W.W.)
- Jena Center for Healthy Ageing, Jena University Hospital, 07747 Jena, Thuringia, Germany
| | - Alexandra Kretz
- Hans-Berger Department of Neurology, Jena University Hospital, 07747 Jena, Thuringia, Germany; (Q.A.); (C.S.); (D.W.); (O.W.W.)
- Jena Center for Healthy Ageing, Jena University Hospital, 07747 Jena, Thuringia, Germany
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22
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Franklin A, Steele EJ, Lindley RA. A proposed reverse transcription mechanism for (CAG)n and similar expandable repeats that cause neurological and other diseases. Heliyon 2020; 6:e03258. [PMID: 32140575 PMCID: PMC7044655 DOI: 10.1016/j.heliyon.2020.e03258] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 09/26/2019] [Accepted: 01/15/2020] [Indexed: 12/12/2022] Open
Abstract
The mechanism of (CAG)n repeat generation, and related expandable repeat diseases in non-dividing cells, is currently understood in terms of a DNA template-based DNA repair synthesis process involving hairpin stabilized slippage, local error-prone repair via MutSβ (MSH2-MSH3) hairpin protective stabilization, then nascent strand extension by DNA polymerases-β and -δ. We advance a very similar slipped hairpin-stabilized model involving MSH2-MSH3 with two key differences: the copying template may also be the nascent pre-mRNA with the repair pathway being mediated by the Y-family error-prone enzymes DNA polymerase-η and DNA polymerase-κ acting as reverse transcriptases. We argue that both DNA-based and RNA-based mechanisms could well be activated in affected non-dividing brain cells in vivo. Here, we compare the advantages of the RNA/RT-based model proposed by us as an adjunct to previously proposed models. In brief, our model depends upon dysregulated innate and adaptive immunity cascades involving AID/APOBEC and ADAR deaminases that are known to be involved in normal locus-specific immunoglobulin somatic hypermutation, cancer progression and somatic mutations at many off-target non-immunoglobulin sites across the genome: we explain how these processes could also play an active role in repeat expansion diseases at RNA polymerase II-transcribed genes.
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Affiliation(s)
- Andrew Franklin
- Medical Department, Novartis Pharmaceuticals UK Limited, 200 Frimley Business Park, Frimley, Surrey, GU16 7SR, United Kingdom
| | - Edward J. Steele
- Melville Analytics Pty Ltd, Melbourne, Vic, 3004, Australia
- CYO’Connor ERADE Village Foundation, Perth, WA, Australia
| | - Robyn A. Lindley
- GMDxgenomics, Melbourne, Vic, Australia
- Department of Clinical Pathology, Faculty of Medicine, Dentistry & Health Sciences, University of Melbourne, Vic, Australia
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23
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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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Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism. PLoS Genet 2019; 15:e1008494. [PMID: 31815930 PMCID: PMC6922473 DOI: 10.1371/journal.pgen.1008494] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Revised: 12/19/2019] [Accepted: 10/25/2019] [Indexed: 12/16/2022] Open
Abstract
Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction. Processes that ensure genome stability are crucial for all organisms to avoid mutations and decrease the risk of diseases. The coordinated activity of mechanisms underlying the maintenance of high-fidelity DNA duplication and repair is critical to deal with the malfunction of replication forks or DNA damage. Repeated sequences in DNA are particularly prone to instability; these sequences undergo expansions or contractions, leading in humans to various neurological, neurodegenerative, and neuromuscular disorders. A mutant form of one of the noncatalytic subunits of active DNA helicase complex impairs DNA replication. Here, we show that this form also significantly increases the instability of mononucleotide, dinucleotide, trinucleotide and longer repeat tracts. Our results suggest that in cells that harbor a mutated variant of the helicase complex, continuation of DNA replication is facilitated by recombination processes, and this mechanism can be highly mutagenic during repair synthesis through repetitive regions, especially regions that form secondary structures. Our results indicate that proper functioning of the DNA helicase complex is crucial for maintenance of the stability of repeated DNA sequences, especially in the context of recently described disorders in which mutations or deregulation of the human homologs of genes encoding DNA helicase subunits were observed.
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Dynamic DNA Energy Landscapes and Substrate Complexity in Triplet Repeat Expansion and DNA Repair. Biomolecules 2019; 9:biom9110709. [PMID: 31698848 PMCID: PMC6920812 DOI: 10.3390/biom9110709] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Revised: 10/29/2019] [Accepted: 10/31/2019] [Indexed: 12/14/2022] Open
Abstract
DNA repeat domains implicated in DNA expansion diseases exhibit complex conformational and energy landscapes that impact biological outcomes. These landscapes include ensembles of entropically driven positional interchanges between isoenergetic, isomeric looped states referred to as rollamers. Here, we present evidence for the position-dependent impact on repeat DNA energy landscapes of an oxidative lesion (8oxodG) and of an abasic site analogue (tetrahydrofuran, F), the universal intermediate in base excision repair (BER). We demonstrate that these lesions modulate repeat bulge loop distributions within the wider dynamic rollamer triplet repeat landscapes. We showed that the presence of a lesion disrupts the energy degeneracy of the rollameric positional isomers. This lesion-induced disruption leads to the redistribution of loop isomers within the repeat loop rollamer ensemble, favoring those rollameric isomers where the lesion is positioned to be energetically least disruptive. These dynamic ensembles create a highly complex energy/conformational landscape of potential BER enzyme substrates to select for processing or to inhibit processing. We discuss the implications of such lesion-induced alterations in repeat DNA energy landscapes in the context of potential BER repair outcomes, thereby providing a biophysical basis for the intriguing in vivo observation of a linkage between pathogenic triplet repeat expansion and DNA repair.
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Murata A, Nakamori M, Nakatani K. Modulating RNA secondary and tertiary structures by mismatch binding ligands. Methods 2019; 167:78-91. [DOI: 10.1016/j.ymeth.2019.05.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2018] [Revised: 05/05/2019] [Accepted: 05/07/2019] [Indexed: 12/21/2022] Open
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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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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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Konopka A, Atkin JD. The Emerging Role of DNA Damage in the Pathogenesis of the C9orf72 Repeat Expansion in Amyotrophic Lateral Sclerosis. Int J Mol Sci 2018; 19:ijms19103137. [PMID: 30322030 PMCID: PMC6213462 DOI: 10.3390/ijms19103137] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2018] [Revised: 10/09/2018] [Accepted: 10/09/2018] [Indexed: 02/07/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal, rapidly progressing neurodegenerative disease affecting motor neurons, and frontotemporal dementia (FTD) is a behavioural disorder resulting in early-onset dementia. Hexanucleotide (G4C2) repeat expansions in the gene encoding chromosome 9 open reading frame 72 (C9orf72) are the major cause of familial forms of both ALS (~40%) and FTD (~20%) worldwide. The C9orf72 repeat expansion is known to form abnormal nuclei acid structures, such as hairpins, G-quadruplexes, and R-loops, which are increasingly associated with human diseases involving microsatellite repeats. These configurations form during normal cellular processes, but if they persist they also damage DNA, and hence are a serious threat to genome integrity. It is unclear how the repeat expansion in C9orf72 causes ALS, but recent evidence implicates DNA damage in neurodegeneration. This may arise from abnormal nucleic acid structures, the greatly expanded C9orf72 RNA, or by repeat-associated non-ATG (RAN) translation, which generates toxic dipeptide repeat proteins. In this review, we detail recent advances implicating DNA damage in C9orf72-ALS. Furthermore, we also discuss increasing evidence that targeting these aberrant C9orf72 confirmations may have therapeutic value for ALS, thus revealing new avenues for drug discovery for this disorder.
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Affiliation(s)
- Anna Konopka
- Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia.
| | - Julie D Atkin
- Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia.
- La Trobe Institute for Molecular Science, Melbourne, VIC 3086, Australia.
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Abstract
Diseases such as Huntington's disease and certain spinocerebellar ataxias are caused by the expansion of genomic cytosine-adenine-guanine (CAG) trinucleotide repeats beyond a specific threshold. These diseases are all characterised by neurological symptoms and central neurodegeneration, but our understanding of how expanded repeats drive neuronal loss is incomplete. Recent human genetic evidence implicates DNA repair pathways, especially mismatch repair, in modifying the onset and progression of CAG repeat diseases. Repair pathways might operate directly on repeat sequences by licensing or inhibiting repeat expansion in neurons. Alternatively, or in addition, because many of the genes containing pathogenic CAG repeats encode proteins that themselves have roles in the DNA damage response, it is possible that repeat expansions impair specific DNA repair pathways. DNA damage could then accrue in neurons, leading to further expansion at repeat loci, thus setting up a vicious cycle of pathology. In this review, we consider DNA damage and repair pathways in postmitotic neurons in the context of disease-causing CAG repeats. Investigating and understanding these pathways, which are clearly relevant in promoting and ameliorating disease in humans, is a research priority, as they are known to modify disease and therefore constitute prevalidated drug targets.
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Affiliation(s)
- Thomas H Massey
- Institute of Psychological Medicine and Clinical Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics, Hadyn Ellis Building, Cardiff University, Cardiff, CF24 4HQ, UK
| | - Lesley Jones
- Institute of Psychological Medicine and Clinical Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics, Hadyn Ellis Building, Cardiff University, Cardiff, CF24 4HQ, UK
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Farg MA, Konopka A, Soo KY, Ito D, Atkin JD. The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis. Hum Mol Genet 2018; 26:2882-2896. [PMID: 28481984 DOI: 10.1093/hmg/ddx170] [Citation(s) in RCA: 114] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2017] [Accepted: 05/02/2017] [Indexed: 12/13/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease affecting motor neurons. Hexanucleotide (GGGGCC) repeat expansions in a non-coding region of C9orf72 are the major cause of familial ALS and frontotemporal dementia (FTD) worldwide. The C9orf72 repeat expansion undergoes repeat-associated non-ATG (RAN) translation to produce five dipeptide repeat proteins (DRPs), including poly(GR) and poly(PR). Whilst it remains unclear how mutations in C9orf72 lead to neurodegeneration in ALS/FTD, dysfunction to the nucleolus and R loop formation are implicated as pathogenic mechanisms. These events can damage DNA and hence genome integrity. Cells activate the DNA damage response (DDR) with the aim of repairing this damage. However, if the damage cannot be repaired, apoptosis is triggered. In lumbar motor neurons from C9orf72-positive ALS patients, we demonstrate significant up-regulation of markers of the DDR compared to controls: phosphorylated histone 2AX (γ-H2AX), phosphorylated ataxia telangiectasia mutated (p-ATM), cleaved poly (ADP-Ribose) polymerase 1 (PARP-1) and tumour suppressor p53-binding protein (53BP1). Similarly, significant up-regulation of γ-H2AX and p-ATM was detected in neuronal cells expressing poly(GR)100 and poly(PR)100 compared to controls, revealing that DNA damage is triggered by the DRPs. Nucleophosmin (NPM1) is a histone chaperone induced during the DDR, which interacts with APE1 to enhance DNA repair. We also demonstrate that more NPM1 precipitates with APE1 in C9orf72 patients compared to controls. Furthermore, overexpression of NPM1 inhibits apoptosis in cells expressing poly(GR)100 and poly(PR)100. This study therefore demonstrates that DNA damage is activated by the C9orf72 repeat expansion in ALS.
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Affiliation(s)
- Manal A Farg
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia
| | - Anna Konopka
- Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, 2 Technology Place, Macquarie University, NSW 2109, Australia
| | - Kai Ying Soo
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia
| | - Daisuke Ito
- Department of Neurology, School of Medicine, Keio University, Tokyo 160-8582, Japan
| | - Julie D Atkin
- Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.,Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, 2 Technology Place, Macquarie University, NSW 2109, Australia
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32
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Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins. Nat Struct Mol Biol 2017; 24:765-777. [PMID: 28805808 DOI: 10.1038/nsmb.3441] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2017] [Accepted: 06/23/2017] [Indexed: 12/21/2022]
Abstract
Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.
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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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Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in Schizosaccharomyces pombe. Genetics 2017; 206:481-496. [PMID: 28292918 DOI: 10.1534/genetics.117.200972] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Accepted: 02/24/2017] [Indexed: 11/18/2022] Open
Abstract
While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in nonhomologous end-joining (NHEJ) is unclear as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mammals have different requirements for repairing cut DNA ends. Most double-strand breaks (DSBs) require nucleolytic processing prior to DNA ligation. Therefore, we studied repair using the Hermes transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single Hermes insertions using a novel S. pombe transient transfection system, and used Hermes excision to show a requirement for MRN in the NHEJ of nonligatable ends. NHEJ repair was indicated by the >1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had <5 bp of sequence loss or mutation, characteristic for NHEJ and similar excision events in metazoans, and in contrast to the more extensive loss seen in S. cerevisiaeS. pombe NHEJ was reduced >1000-fold in cells lacking each MRN subunit, and loss of MRN-associated Ctp1 caused a 30-fold reduction. An Mre11 dimer is thought to hold DNA ends together for repair, and Mre11 dimerization domain mutations reduced repair 300-fold. In contrast, a mre11 mutant defective in endonucleolytic activity, the same mutant lacking Ctp1, or the triple mutant also lacking the putative hairpin nuclease Pso2 showed wild-type levels of repair. Thus, MRN may act to recruit the hairpin opening activity that allows subsequent repair.
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Ueki J, Nakamori M, Nakamura M, Nishikawa M, Yoshida Y, Tanaka A, Morizane A, Kamon M, Araki T, Takahashi MP, Watanabe A, Inagaki N, Sakurai H. Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability. Sci Rep 2017; 7:42522. [PMID: 28211918 PMCID: PMC5304155 DOI: 10.1038/srep42522] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2016] [Accepted: 01/09/2017] [Indexed: 02/08/2023] Open
Abstract
Myotonic dystrophy type 1 (DM1) is an autosomal-dominant multi-system disease caused by expanded CTG repeats in dystrophia myotonica protein kinase (DMPK). The expanded CTG repeats are unstable and can increase the length of the gene with age, which worsens the symptoms. In order to establish a human stem cell system suitable for the investigation of repeat instability, DM1 patient-derived iPSCs were generated and differentiated into three cell types commonly affected in DM1, namely cardiomyocytes, neurons and myocytes. Then we precisely analysed the CTG repeat lengths in these cells. Our DM1-iPSCs showed a gradual lengthening of CTG repeats with unchanged repeat distribution in all cell lines depending on the passage numbers of undifferentiated cells. However, the average CTG repeat length did not change significantly after differentiation into different somatic cell types. We also evaluated the chromatin accessibility in DM1-iPSCs using ATAC-seq. The chromatin status in DM1 cardiomyocytes was closed at the DMPK locus as well as at SIX5 and its promoter region, whereas it was open in control, suggesting that the epigenetic modifications may be related to the CTG repeat expansion in DM1. These findings may help clarify the role of repeat instability in the CTG repeat expansion in DM1.
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Affiliation(s)
- Junko Ueki
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.,Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Masayuki Nakamori
- Department of Neurology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Masahiro Nakamura
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Misato Nishikawa
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Yoshinori Yoshida
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Azusa Tanaka
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Asuka Morizane
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Masayoshi Kamon
- Department of Peripheral Nervous System Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan
| | - Toshiyuki Araki
- Department of Peripheral Nervous System Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan
| | - Masanori P Takahashi
- Department of Neurology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.,Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Akira Watanabe
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Nobuya Inagaki
- Department of Diabetes, Endocrinology and Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
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Guo P, Lam SL. Unusual structures of CCTG repeats and their participation in repeat expansion. Biomol Concepts 2016; 7:331-340. [DOI: 10.1515/bmc-2016-0024] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2016] [Accepted: 11/01/2016] [Indexed: 11/15/2022] Open
Abstract
AbstractCCTG repeat expansion in intron 1 of the cellular nucleic acid-binding protein (CNBP) gene has been identified to be the genetic cause of myotonic dystrophy type 2 (DM2). Yet the underlying reasons for the genetic instability in CCTG repeats remain elusive. In recent years, CCTG repeats have been found to form various types of unusual secondary structures including mini-dumbbell (MDB), hairpin and dumbbell, revealing that there is a high structural diversity in CCTG repeats intrinsically. Upon strand slippage, the formation of unusual structures in the nascent strand during DNA replication has been proposed to be the culprit of CCTG repeat expansions. On the one hand, the thermodynamic stability, size, and conformational dynamics of these unusual structures affect the propensity of strand slippage. On the other hand, these structural properties determine whether the unusual structure can successfully escape from DNA repair. In this short overview, we first summarize the recent advances in elucidating the solution structures of CCTG repeats. We then discuss the potential pathways by which these unusual structures bring about variable sizes of repeat expansion, high strand slippage propensity and efficient repair escape.
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Affiliation(s)
- Pei Guo
- 1Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
| | - Sik Lok Lam
- 1Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
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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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38
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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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39
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Lai Y, Budworth H, Beaver JM, Chan NLS, Zhang Z, McMurray CT, Liu Y. Crosstalk between MSH2-MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair. Nat Commun 2016; 7:12465. [PMID: 27546332 PMCID: PMC4996945 DOI: 10.1038/ncomms12465] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2016] [Accepted: 07/06/2016] [Indexed: 01/07/2023] Open
Abstract
Studies in knockout mice provide evidence that MSH2-MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2-MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2-MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.
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Affiliation(s)
- Yanhao Lai
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, Florida 33199, USA
| | - Helen Budworth
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 33R249, Berkeley, California 94720, USA
| | - Jill M. Beaver
- Biochemistry Ph.D. Program, Florida International University, 11200 SW 8th Street, Miami, Florida 33199, USA
| | - Nelson L. S. Chan
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 33R249, Berkeley, California 94720, USA
| | - Zunzhen Zhang
- Department of Occupational and Environmental Health, Sichuan University West China School of Public Health, 16#, Section 3, Renmin Nan Lu, Chengdu, Sichuan 610041, China
| | - Cynthia T. McMurray
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, 33R249, Berkeley, California 94720, USA
| | - Yuan Liu
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, Florida 33199, USA
- Biochemistry Ph.D. Program, Florida International University, 11200 SW 8th Street, Miami, Florida 33199, USA
- Biomolecular Sciences Institute, School of Integrated Sciences and Humanity, Florida International University, 11200 SW 8th Street, Miami, Florida 33199, USA
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40
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Shibata T, Nakatani K. Fluorescence Probe for Detecting CCG Trinucleotide Repeat DNA Expansion and Slip-Out. Chembiochem 2016; 17:1685-8. [DOI: 10.1002/cbic.201600200] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Indexed: 11/09/2022]
Affiliation(s)
- Tomonori Shibata
- Regulatory Bioorganic Chemistry; The Institute of Scientific and Industrial Research; Osaka University; 8-1, Mihogaoka Ibaraki 567-0047 Japan
| | - Kazuhiko Nakatani
- Regulatory Bioorganic Chemistry; The Institute of Scientific and Industrial Research; Osaka University; 8-1, Mihogaoka Ibaraki 567-0047 Japan
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41
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Guo J, Gu L, Leffak M, Li GM. MutSβ promotes trinucleotide repeat expansion by recruiting DNA polymerase β to nascent (CAG)n or (CTG)n hairpins for error-prone DNA synthesis. Cell Res 2016; 26:775-86. [PMID: 27255792 PMCID: PMC5129881 DOI: 10.1038/cr.2016.66] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Revised: 05/15/2016] [Accepted: 05/17/2016] [Indexed: 12/12/2022] Open
Abstract
Expansion of (CAG)•(CTG) repeats causes a number of familial neurodegenerative disorders. Although the underlying mechanism remains largely unknown, components involved in DNA mismatch repair, particularly mismatch recognition protein MutSβ (a MSH2-MSH3 heterodimer), are implicated in (CAG)•(CTG) repeat expansion. In addition to recognizing small insertion-deletion loop-outs, MutSβ also specifically binds DNA hairpin imperfect heteroduplexes formed within (CAG)n•(CTG)n sequences. However, whether or not and how MutSβ binding triggers expansion of (CAG)•(CTG) repeats remain unknown. We show here that purified recombinant MutSβ physically interacts with DNA polymerase β (Polβ) and stimulates Polβ-catalyzed (CAG)n or (CTG)n hairpin retention. Consistent with these in vitro observations, MutSβ and Polβ interact with each other in vivo, and colocalize at (CAG)•(CTG) repeats during DNA replication. Our data support a model for error-prone processing of (CAG)n or (CTG)n hairpins by MutSβ and Polβ during DNA replication and/or repair: MutSβ recognizes (CAG)n or (CTG)n hairpins formed in the nascent DNA strand, and recruits Polβ to the complex, which then utilizes the hairpin as a primer for extension, leading to (CAG)•(CTG) repeat expansion. This study provides a novel mechanism for trinucleotide repeat expansion in both dividing and non-dividing cells.
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Affiliation(s)
- Jinzhen Guo
- Department of Basic Medical Sciences, Tsinghua University School of Medicine, Beijing 100084, China.,Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1450 Biggy Street, Los Angeles, CA 90033, USA
| | - Liya Gu
- Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1450 Biggy Street, Los Angeles, CA 90033, USA
| | - Michael Leffak
- Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA
| | - Guo-Min Li
- Department of Basic Medical Sciences, Tsinghua University School of Medicine, Beijing 100084, China.,Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1450 Biggy Street, Los Angeles, CA 90033, USA
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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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Souza GN, Kersting N, Krum-Santos AC, Santos ASP, Furtado GV, Pacheco D, Gonçalves TA, Saute JA, Schuler-Faccini L, Mattos EP, Saraiva-Pereira ML, Jardim LB. Spinocerebellar ataxia type 3/Machado-Joseph disease: segregation patterns and factors influencing instability of expanded CAG transmissions. Clin Genet 2016; 90:134-40. [PMID: 26693702 DOI: 10.1111/cge.12719] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2015] [Revised: 12/16/2015] [Accepted: 12/17/2015] [Indexed: 11/30/2022]
Abstract
Controversies about Mendelian segregation and CAG expansion (CAGexp) instabilities during meiosis in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) need clarification. Additional evidence about these issues was obtained from the cohort of all SCA3/MJD individuals living in South Brazil. A survey was carried out to update information registered since 2001. Deaths were checked with the Public Information System, and data was made anonymous. Anticipation and delta-CAGexp from parent-offspring pairs, and delta-CAGexp between siblings were obtained. One hundred and fifty-nine families (94% of the entire registry) were retrieved, comprising 3725 living individuals as of 2015, 625 of these being symptomatic. Minimal prevalence was 6:100,000. Carriers of a CAGexp represented 65.6% of sibs in the genotyped offspring (p < 0.001). Median instability was larger among paternal than maternal transmissions, and instabilities correlated with anticipation (r = 0.38; p = 0.001). Age of the parent correlated to delta-CAGexp among 115 direct parent-offspring CAGexp transmissions (ρ = 0.23, p = 0.014). In 98 additional kindreds, the delta-CAGexp between 269 siblings correlated with their delta-of-age (ρ = 0.27, p < 0.0001). SCA3/MJD was associated with a segregation distortion favoring the expanded allele in our cohort. Instability of expansion during meiosis was weakly influenced by the age of the transmitting parent at the time of conception.
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Affiliation(s)
- G N Souza
- Programa de Pós-Graduação em Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - N Kersting
- Programa de Pós-Graduação em Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - A C Krum-Santos
- Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - A S P Santos
- Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - G V Furtado
- Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Laboratório de Identificação Genética, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - D Pacheco
- Faculdade de Serviço Social, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - T A Gonçalves
- Escola de Enfermagem, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - J A Saute
- Laboratório de Identificação Genética, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.,Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - L Schuler-Faccini
- Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.,Departamento de Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Instituto de Genética Médica Populacional (INAGEMP), Porto Alegre, Brazil
| | - E P Mattos
- Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Laboratório de Identificação Genética, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
| | - M L Saraiva-Pereira
- Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Laboratório de Identificação Genética, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.,Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.,Instituto de Genética Médica Populacional (INAGEMP), Porto Alegre, Brazil.,Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
| | - L B Jardim
- Programa de Pós-Graduação em Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.,Laboratório de Identificação Genética, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.,Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.,Instituto de Genética Médica Populacional (INAGEMP), Porto Alegre, Brazil.,Departamento de Medicina Interna, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
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44
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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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45
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Kansal R, Li X, Shen J, Samuel D, Laningham F, Lee H, Panigrahi GB, Shuen A, Kantarci S, Dorrani N, Reiss J, Shintaku P, Deignan JL, Strom SP, Pearson CE, Vilain E, Grody WW. An infant withMLH3variants,FOXG1-duplication and multiple, benign cranial and spinal tumors: A clinical exome sequencing study. Genes Chromosomes Cancer 2015; 55:131-42. [DOI: 10.1002/gcc.22319] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2014] [Revised: 08/13/2015] [Accepted: 08/14/2015] [Indexed: 12/27/2022] Open
Affiliation(s)
- Rina Kansal
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Xinmin Li
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Joseph Shen
- Medical Genetics and Metabolism; Valley Children's Hospital; Madera CA 93636
| | - David Samuel
- Hematology/Oncology, Valley Children's Hospital; Madera CA 93636
| | - Fred Laningham
- Department of Radiology; Valley Children's Hospital; Madera CA 93636
| | - Hane Lee
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Gagan B. Panigrahi
- Program of Genetics & Genome Biology; The Hospital for Sick Children, Peter Gilgan Center for Research and Learning; Toronto Ontario MSG 0A4 Canada
| | - Andrew Shuen
- Program of Genetics & Genome Biology; The Hospital for Sick Children, Peter Gilgan Center for Research and Learning; Toronto Ontario MSG 0A4 Canada
- Program of Molecular Genetics, University of Toronto; Toronto, Ontario M5S 1A1 Canada
| | - Sibel Kantarci
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Naghmeh Dorrani
- Pediatrics, University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Jean Reiss
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Peter Shintaku
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Joshua L. Deignan
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Samuel P. Strom
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Christopher E. Pearson
- Program of Genetics & Genome Biology; The Hospital for Sick Children, Peter Gilgan Center for Research and Learning; Toronto Ontario MSG 0A4 Canada
- Program of Molecular Genetics, University of Toronto; Toronto, Ontario M5S 1A1 Canada
| | - Eric Vilain
- Pediatrics, University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
- Human Genetics, University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
| | - Wayne W. Grody
- Pathology and Laboratory Medicine; University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
- Pediatrics, University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
- Human Genetics, University of California at Los Angeles, David Geffen School of Medicine; Los Angeles CA 90095
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46
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Gerhardt J. Epigenetic modifications in human fragile X pluripotent stem cells; Implications in fragile X syndrome modeling. Brain Res 2015; 1656:55-62. [PMID: 26475977 DOI: 10.1016/j.brainres.2015.10.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Revised: 08/18/2015] [Accepted: 10/02/2015] [Indexed: 12/18/2022]
Abstract
Patients with fragile X syndrome (FXS) exhibit moderate to severe intellectual disabilities. In addition, one-third of FXS patients show characteristics of autism spectrum disorder. FXS is caused by a trinucleotide repeat expansion, which leads to silencing of the fragile X mental retardation (FMR1) gene. The absence of the FMR1 gene product, FMRP, is the reason for the disease symptoms. It has been suggested that repeat instability and transcription of the FMR1 gene occur during early embryonic development, while after cell differentiation repeats become stable and the FMR1 gene is silent. Epigenetic marks, such as DNA methylation, are associated with gene silencing and repeat stability at the FMR1 locus. However, the mechanisms leading to gene silencing and repeat expansion are still ambiguous, because studies at the human genomic locus were limited until now. The FXS pluripotent stem cells, recently derived from FXS adult cells and FXS blastocysts, are new useful tools to examine these mechanisms at the human endogenous FMR1 locus. This review summarizes the epigenetic features and experimental studies of FXS human embryonic and FXS induced pluripotent stem cells, generated so far. This article is part of a Special Issue entitled SI: Exploiting human neurons.
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Affiliation(s)
- Jeannine Gerhardt
- Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx 10461, USA.
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47
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Guo P, Lam SL. New insights into the genetic instability in CCTG repeats. FEBS Lett 2015; 589:3058-63. [PMID: 26384951 DOI: 10.1016/j.febslet.2015.09.007] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2015] [Revised: 08/18/2015] [Accepted: 09/08/2015] [Indexed: 11/29/2022]
Abstract
Tetranucleotide CCTG repeat expansion is associated with myotonic dystrophy type 2, which is an inherited and progressive muscle degeneration disease. Yet, no cure is available and the molecular mechanism of repeat expansion remains elusive. In this study, we used high-resolution nuclear magnetic resonance spectroscopy to reveal a mini-dumbbell structure formed by two CCTG repeats. Upon slippage in the nascent strand during DNA replication, the formation of the mini-dumbbell provides a possible pathway for a two-repeat expansion. In addition, fast exchange between two competing mini-dumbbells among three repeats results in a mini-loop structure that accounts for one-repeat expansion. These mini-dumbbell and mini-loop intermediates can also co-exist at multiple sites in CCTG repeats, leading to three or larger size repeat expansions.
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Affiliation(s)
- Pei Guo
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
| | - Sik Lok Lam
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
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48
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Williams GM, Surtees JA. MSH3 Promotes Dynamic Behavior of Trinucleotide Repeat Tracts In Vivo. Genetics 2015; 200:737-54. [PMID: 25969461 PMCID: PMC4512540 DOI: 10.1534/genetics.115.177303] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2015] [Accepted: 05/04/2015] [Indexed: 11/18/2022] Open
Abstract
Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease, yet the pathway to expansion remains poorly understood. An important step in expansion is the shift from a stable TNR sequence to an unstable, expanding tract, which is thought to occur once a TNR attains a threshold length. Modeling of human data has indicated that TNR tracts are increasingly likely to expand as they increase in size and to do so in increments that are smaller than the repeat itself, but this has not been tested experimentally. Genetic work has implicated the mismatch repair factor MSH3 in promoting expansions. Using Saccharomyces cerevisiae as a model for CAG and CTG tract dynamics, we examined individual threshold-length TNR tracts in vivo over time in MSH3 and msh3Δ backgrounds. We demonstrate, for the first time, that these TNR tracts are highly dynamic. Furthermore, we establish that once such a tract has expanded by even a few repeat units, it is significantly more likely to expand again. Finally, we show that threshold- length TNR sequences readily accumulate net incremental expansions over time through a series of small expansion and contraction events. Importantly, the tracts were substantially stabilized in the msh3Δ background, with a bias toward contractions, indicating that Msh2-Msh3 plays an important role in shifting the expansion-contraction equilibrium toward expansion in the early stages of TNR tract expansion.
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Affiliation(s)
- Gregory M Williams
- Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, New York 14214
| | - Jennifer A Surtees
- Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, New York 14214 Genetics, Genomics and Bioinformatics Program, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, New York 14214
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49
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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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Barros SA, Chenoweth DM. Triptycene-based small molecules modulate (CAG)·(CTG) repeat junctions. Chem Sci 2015; 6:4752-4755. [PMID: 26366282 PMCID: PMC4538686 DOI: 10.1039/c5sc01595b] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2015] [Accepted: 05/27/2015] [Indexed: 01/19/2023] Open
Abstract
A triptycene-based scaffold is used to develop a new class of ligands for modulating the structure of junction forming trinucleotide repeat expansion sequences.
Nucleic acid three-way junctions (3WJs) play key roles in biological processes such as nucleic acid replication in addition to being implicated as dynamic transient intermediates in trinucleotide repeat sequences. Structural modulation of specific nucleic acid junctions could allow for control of biological processes and disease states at the nucleic acid level. Trinucleotide repeat expansions are associated with several neurodegenerative diseases where dynamic slippage is thought to occur during replication, forming transient 3WJ intermediates with the complementary strand. Here, we report triptycene-based molecules that bind to a d(CAG)·(CTG) repeat using a gel shift assay, fluorescence-quenching and circular dichroism.
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Affiliation(s)
- Stephanie A Barros
- Department of Chemistry , University of Pennsylvania , 231 South 34th Street , Philadelphia , PA 19104-6323 , USA .
| | - David M Chenoweth
- Department of Chemistry , University of Pennsylvania , 231 South 34th Street , Philadelphia , PA 19104-6323 , USA .
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