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Zachayus A, Loup-Forest J, Cura V, Poterszman A. Nucleotide Excision Repair: Insights into Canonical and Emerging Functions of the Transcription/DNA Repair Factor TFIIH. Genes (Basel) 2025; 16:231. [PMID: 40004560 PMCID: PMC11855273 DOI: 10.3390/genes16020231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2025] [Revised: 01/31/2025] [Accepted: 02/06/2025] [Indexed: 02/27/2025] Open
Abstract
Nucleotide excision repair (NER) is a universal cut-and-paste DNA repair mechanism that corrects bulky DNA lesions such as those caused by UV radiation, environmental mutagens, and some chemotherapy drugs. In this review, we focus on the human transcription/DNA repair factor TFIIH, a key player of the NER pathway in eukaryotes. This 10-subunit multiprotein complex notably verifies the presence of a lesion and opens the DNA around the damage via its XPB and XPD subunits, two proteins identified in patients suffering from Xeroderma Pigmentosum syndrome. Isolated as a class II gene transcription factor in the late 1980s, TFIIH is a prototypic molecular machine that plays an essential role in both DNA repair and transcription initiation and harbors a DNA helicase, a DNA translocase, and kinase activity. More recently, TFIIH subunits have been identified as participating in other cellular processes, including chromosome segregation during mitosis, maintenance of mitochondrial DNA integrity, and telomere replication.
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Affiliation(s)
- Amélie Zachayus
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
| | - Jules Loup-Forest
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
| | - Vincent Cura
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
| | - Arnaud Poterszman
- Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France; (A.Z.); (J.L.-F.); (V.C.)
- Centre National de la Recherche Scientifique (CNRS), UMR 7104, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Institut National De La Sante et de la Recherche Médicale (Inserm), UMR S 1258, 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
- Equipe Labellisée Ligue Contre le Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67400 Illkirch-Graffenstaden, France
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Guo Y, Li P, Zhang J, Hao S, Zhou X, Di C, Long F, Zhang H, Si J. Carbon ion irradiation conquers the radioresistance by inducing complex DNA damage and apoptosis in U251 human glioblastomas cells. Med Oncol 2025; 42:64. [PMID: 39903402 DOI: 10.1007/s12032-025-02616-5] [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: 11/01/2024] [Accepted: 01/22/2025] [Indexed: 02/06/2025]
Abstract
Glioblastoma multiforme (GBM) is the most malignant brain tumor, with radiotherapy frequently employed following surgical resection. However, conventional radiation therapies often yield suboptimal results. This study investigated the effects of X-ray and carbon ion irradiation on the glioblastoma cell line U251 to assess the distinctive advantages of carbon ion treatment and explore mechanisms for overcoming radiation resistance. The findings indicated that carbon ion irradiation more effectively inhibited colony formation and induced more severe apoptosis and cell cycle disorder in U251 cells. Immunofluorescence assays revealed larger and more abundant ϒ-H2AX and 53BP1 foci in the carbon ion irradiation group. Western blot analysis demonstrated that carbon ion-induced DNA damage repair involved a complex array of pathways, with the RAD51-mediated homologous recombination (HR) pathway being predominant, while the Rad23B-mediated nucleotide excision repair (NER) pathway and XRCC1-mediated base excision repair (BER) were more relevant in response to X-ray irradiation. These results suggest that carbon ion irradiation may overcome radioresistance by inducing more complex DNA damage and apoptosis, thus providing insights for targeting new strategies in combining gene therapy with radiotherapy.
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Affiliation(s)
- Yulu Guo
- Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou, 730000, Gansu, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
| | - Pingping Li
- Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou, 730000, Gansu, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
| | - Jinhua Zhang
- Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou, 730000, Gansu, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
| | - Sijia Hao
- Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou, 730000, Gansu, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
| | - Xuan Zhou
- School of Basic Medicine, Gansu University of Chinese Medicine, Lanzhou, China
| | - Cuixia Di
- Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou, 730000, Gansu, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, China
| | - Feng Long
- School of Basic Medicine, Gansu University of Chinese Medicine, Lanzhou, China
| | - Hong Zhang
- Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou, 730000, Gansu, China.
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, China.
| | - Jing Si
- Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou, 730000, Gansu, China.
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, China.
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Mevissen TET, Kümmecke M, Schmid EW, Farnung L, Walter JC. STK19 positions TFIIH for cell-free transcription-coupled DNA repair. Cell 2024; 187:7091-7106.e24. [PMID: 39547228 PMCID: PMC11645862 DOI: 10.1016/j.cell.2024.10.020] [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: 05/29/2024] [Revised: 08/29/2024] [Accepted: 10/12/2024] [Indexed: 11/17/2024]
Abstract
In transcription-coupled nucleotide excision repair (TC-NER), stalled RNA polymerase II (RNA Pol II) binds CSB and CRL4CSA, which cooperate with UVSSA and ELOF1 to recruit TFIIH. To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also requires STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9-Å cryo-electron microscopy structure shows that STK19 binds the TC-NER complex through CSA and the RPB1 subunit of RNA Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of RNA Pol II for lesion verification. Our analysis of cell-free TC-NER suggests that STK19 couples RNA Pol II stalling to downstream repair events.
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Affiliation(s)
- Tycho E T Mevissen
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA, USA
| | - Maximilian Kümmecke
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Ernst W Schmid
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Lucas Farnung
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
| | - Johannes C Walter
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA, USA.
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Mevissen TE, Kümmecke M, Schmid EW, Farnung L, Walter JC. STK19 positions TFIIH for cell-free transcription-coupled DNA repair. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.22.604623. [PMID: 39091863 PMCID: PMC11291053 DOI: 10.1101/2024.07.22.604623] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 08/04/2024]
Abstract
In transcription-coupled repair, stalled RNA polymerase II (Pol II) is recognized by CSB and CRL4CSA, which co-operate with UVSSSA and ELOF1 to recruit TFIIH for nucleotide excision repair (TC-NER). To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also depends on STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9 Å cryo-electron microscopy structure shows that STK19 joins the TC-NER complex by binding CSA and the RPB1 subunit of Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of Pol II for lesion verification. In summary, our analysis of cell-free TC-NER suggests that STK19 couples RNA polymerase II stalling to downstream repair events.
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Affiliation(s)
- Tycho E.T. Mevissen
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- Howard Hughes Medical Institute
| | - Maximilian Kümmecke
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Ernst W. Schmid
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Lucas Farnung
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Johannes C. Walter
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- Howard Hughes Medical Institute
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Gong W, Holmberg H, Lu C, Huang M, Li S. Interplay of the Tfb1 pleckstrin homology domain with Rad2 and Rad4 in transcription coupled and global genomic nucleotide excision repair. Nucleic Acids Res 2024; 52:6333-6346. [PMID: 38634797 PMCID: PMC11194066 DOI: 10.1093/nar/gkae286] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Revised: 03/29/2024] [Accepted: 04/04/2024] [Indexed: 04/19/2024] Open
Abstract
Transcription-coupled repair (TCR) and global genomic repair (GGR) are two subpathways of nucleotide excision repair (NER). The TFIIH subunit Tfb1 contains a Pleckstrin homology domain (PHD), which was shown to interact with one PHD-binding segment (PB) of Rad4 and two PHD-binding segments (PB1 and PB2) of Rad2 in vitro. Whether and how the different Rad2 and Rad4 PBs interact with the same Tfb1 PHD, and whether and how they affect TCR and GGR within the cell remain mysterious. We found that Rad4 PB constitutively interacts with Tfb1 PHD, and the two proteins may function within one module for damage recognition in TCR and GGR. Rad2 PB1 protects Tfb1 from degradation and interacts with Tfb1 PHD at a basal level, presumably within transcription preinitiation complexes when NER is inactive. During a late step of NER, the interaction between Rad2 PB1 and Tfb1 PHD augments, enabling efficient TCR and GGR. Rather than interacting with Tfb1 PHD, Rad2 PB2 constrains the basal interaction between Rad2 PB1 and Tfb1 PHD, thereby weakening the protection of Tfb1 from degradation and enabling rapid augmentation of their interactions within TCR and GGR complexes. Our results shed new light on NER mechanisms.
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Affiliation(s)
- Wenzhi Gong
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Hannah Holmberg
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Cheng Lu
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Michelle Huang
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Shisheng Li
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
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Kose C, Lindsey-Boltz LA, Sancar A, Jiang Y. Genome-wide analysis of transcription-coupled repair reveals novel transcription events in Caenorhabditis elegans. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.10.12.562083. [PMID: 37904932 PMCID: PMC10614815 DOI: 10.1101/2023.10.12.562083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/01/2023]
Abstract
Bulky DNA adducts such as those induced by ultraviolet light are removed from the genomes of multicellular organisms by nucleotide excision repair, which occurs through two distinct mechanisms, global repair, requiring the DNA damage recognition-factor XPC (xeroderma pigmentosum complementation group C), and transcription-coupled repair (TCR), which does not. TCR is initiated when elongating RNA polymerase II encounters DNA damage, and thus analysis of genome-wide excision repair in XPC-mutants only repairing by TCR provides a unique opportunity to map transcription events missed by methods dependent on capturing RNA transcription products and thus limited by their stability and/or modifications (5'-capping or 3'-polyadenylation). Here, we have performed the eXcision Repair-sequencing (XR-seq) in the model organism Caenorhabditis elegans to generate genome-wide repair maps from a wild-type strain with normal excision repair, a strain lacking TCR (csb-1), or one that only repairs by TCR (xpc-1). Analysis of the intersections between the xpc-1 XR-seq repair maps with RNA-mapping datasets (RNA-seq, long- and short-capped RNA-seq) reveal previously unrecognized sites of transcription and further enhance our understanding of the genome of this important model organism.
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Affiliation(s)
- Cansu Kose
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Laura A. Lindsey-Boltz
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Aziz Sancar
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Yuchao Jiang
- Department of Statistics, College of Arts and Sciences, Texas A&M University, College Station, TX 77843, USA
- Department of Biology, College of Arts and Sciences, Texas A&M University, College Station, TX 77843
- Department of Biomedical Engineering, College of Engineering, Texas A&M University, College Station, TX 77843
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Kose C, Cao X, Dewey EB, Malkoç M, Adebali O, Sekelsky J, Lindsey-Boltz LA, Sancar A. Cross-species investigation into the requirement of XPA for nucleotide excision repair. Nucleic Acids Res 2024; 52:677-689. [PMID: 37994737 PMCID: PMC10810185 DOI: 10.1093/nar/gkad1104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2023] [Revised: 10/25/2023] [Accepted: 11/01/2023] [Indexed: 11/24/2023] Open
Abstract
After reconstitution of nucleotide excision repair (excision repair) with XPA, RPA, XPC, TFIIH, XPF-ERCC1 and XPG, it was concluded that these six factors are the minimal essential components of the excision repair machinery. All six factors are highly conserved across diverse organisms spanning yeast to humans, yet no identifiable homolog of the XPA gene exists in many eukaryotes including green plants. Nevertheless, excision repair is reported to be robust in the XPA-lacking organism, Arabidopsis thaliana, which raises a fundamental question of whether excision repair could occur without XPA in other organisms. Here, we performed a phylogenetic analysis of XPA across all species with annotated genomes and then quantitatively measured excision repair in the absence of XPA using the sensitive whole-genome qXR-Seq method in human cell lines and two model organisms, Caenorhabditis elegans and Drosophila melanogaster. We find that although the absence of XPA results in inefficient excision repair and UV-sensitivity in humans, flies, and worms, excision repair of UV-induced DNA damage is detectable over background. These studies have yielded a significant discovery regarding the evolution of XPA protein and its mechanistic role in nucleotide excision repair.
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Affiliation(s)
- Cansu Kose
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Xuemei Cao
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Evan B Dewey
- Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Mustafa Malkoç
- Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Türkiye
| | - Ogün Adebali
- Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Türkiye
- Department of Computational Science-Biological Sciences, TÜBITAK Research Institute for Fundamental Sciences, Gebze, Türkiye
| | - Jeff Sekelsky
- Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Laura A Lindsey-Boltz
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Aziz Sancar
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
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Cao X, Kose C, Selby CP, Sancar A. In vitro DNA repair genomics using XR-seq with Escherichia coli and mammalian cell-free extracts. Proc Natl Acad Sci U S A 2023; 120:e2314233120. [PMID: 37844222 PMCID: PMC10614213 DOI: 10.1073/pnas.2314233120] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2023] [Accepted: 09/16/2023] [Indexed: 10/18/2023] Open
Abstract
The XR-seq (eXcision Repair-sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell-free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light-irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell-free extracts and by mammalian cell-free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair.
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Affiliation(s)
- Xuemei Cao
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC27599
| | - Cansu Kose
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC27599
| | - Christopher P. Selby
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC27599
| | - Aziz Sancar
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC27599
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Cohen Y, Adar S. Novel insights into bulky DNA damage formation and nucleotide excision repair from high-resolution genomics. DNA Repair (Amst) 2023; 130:103549. [PMID: 37566959 DOI: 10.1016/j.dnarep.2023.103549] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 07/27/2023] [Accepted: 07/28/2023] [Indexed: 08/13/2023]
Abstract
DNA damages compromise cell function and fate. Cells of all organisms activate a global DNA damage response that includes a signaling stress response, activation of checkpoints, and recruitment of repair enzymes. Especially deleterious are bulky, helix-distorting damages that block transcription and replication. Due to their miscoding nature, these damages lead to mutations and cancer. In human cells, bulky DNA damages are repaired by nucleotide excision repair (NER). To date, the basic mechanism of NER in naked DNA is well defined. Still, there is a fundamental gap in our understanding of how repair is orchestrated despite the packaging of DNA in chromatin, and how it is coordinated with active transcription and replication. The last decade has brought forth huge advances in our ability to detect and assay bulky DNA damages and their repair at single nucleotide resolution across the human genome. Here we review recent findings on the effect of chromatin and DNA-binding proteins on the formation of bulky DNA damages, and novel insights on NER, provided by the recent application of genomic methods.
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Affiliation(s)
- Yuval Cohen
- Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel Canada, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9112102, Israel
| | - Sheera Adar
- Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel Canada, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9112102, Israel.
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Briguglio S, Cambria C, Albizzati E, Marcello E, Provenzano G, Frasca A, Antonucci F. New Views of the DNA Repair Protein Ataxia-Telangiectasia Mutated in Central Neurons: Contribution in Synaptic Dysfunctions of Neurodevelopmental and Neurodegenerative Diseases. Cells 2023; 12:2181. [PMID: 37681912 PMCID: PMC10486624 DOI: 10.3390/cells12172181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Revised: 08/18/2023] [Accepted: 08/27/2023] [Indexed: 09/09/2023] Open
Abstract
Ataxia-Telangiectasia Mutated (ATM) is a serine/threonine protein kinase principally known to orchestrate DNA repair processes upon DNA double-strand breaks (DSBs). Mutations in the Atm gene lead to Ataxia-Telangiectasia (AT), a recessive disorder characterized by ataxic movements consequent to cerebellar atrophy or dysfunction, along with immune alterations, genomic instability, and predisposition to cancer. AT patients show variable phenotypes ranging from neurologic abnormalities and cognitive impairments to more recently described neuropsychiatric features pointing to symptoms hardly ascribable to the canonical functions of ATM in DNA damage response (DDR). Indeed, evidence suggests that cognitive abilities rely on the proper functioning of DSB machinery and specific synaptic changes in central neurons of ATM-deficient mice unveiled unexpected roles of ATM at the synapse. Thus, in the present review, upon a brief recall of DNA damage responses, we focus our attention on the role of ATM in neuronal physiology and pathology and we discuss recent findings showing structural and functional changes in hippocampal and cortical synapses of AT mouse models. Collectively, a deeper knowledge of ATM-dependent mechanisms in neurons is necessary not only for a better comprehension of AT neurological phenotypes, but also for a higher understanding of the pathological mechanisms in neurodevelopmental and degenerative disorders involving ATM dysfunctions.
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Affiliation(s)
- Sabrina Briguglio
- Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Via F.lli Cervi 93, 20129 Segrate (MI) and via Vanvitelli 32, 20129 Milan, MI, Italy; (S.B.); (C.C.); (A.F.)
| | - Clara Cambria
- Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Via F.lli Cervi 93, 20129 Segrate (MI) and via Vanvitelli 32, 20129 Milan, MI, Italy; (S.B.); (C.C.); (A.F.)
| | - Elena Albizzati
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA;
| | - Elena Marcello
- Department of Pharmacological and Biomolecular Sciences, University of Milan, Via Giuseppe Balzaretti 9, 20133 Milan, MI, Italy;
| | - Giovanni Provenzano
- Department of Cellular, Computational and Integrative Biology—CIBIO, University of Trento, Via Sommarive 9, 38068 Trento, TN, Italy;
| | - Angelisa Frasca
- Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Via F.lli Cervi 93, 20129 Segrate (MI) and via Vanvitelli 32, 20129 Milan, MI, Italy; (S.B.); (C.C.); (A.F.)
| | - Flavia Antonucci
- Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Via F.lli Cervi 93, 20129 Segrate (MI) and via Vanvitelli 32, 20129 Milan, MI, Italy; (S.B.); (C.C.); (A.F.)
- Institute of Neuroscience, IN-CNR, Via Raoul Follereau 3, 20854 Vedano al Lambro, MB, Italy
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Lindsey-Boltz LA, Yang Y, Kose C, Deger N, Eynullazada K, Kawara H, Sancar A. Nucleotide excision repair in Human cell lines lacking both XPC and CSB proteins. Nucleic Acids Res 2023; 51:6238-6245. [PMID: 37144462 PMCID: PMC10325923 DOI: 10.1093/nar/gkad334] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2023] [Revised: 04/12/2023] [Accepted: 04/20/2023] [Indexed: 05/06/2023] Open
Abstract
Nucleotide excision repair removes UV-induced DNA damage through two distinct sub-pathways, global repair and transcription-coupled repair (TCR). Numerous studies have shown that in human and other mammalian cell lines that the XPC protein is required for repair of DNA damage from nontranscribed DNA via global repair and the CSB protein is required for repair of lesions from transcribed DNA via TCR. Therefore, it is generally assumed that abrogating both sub-pathways with an XPC-/-/CSB-/- double mutant would eliminate all nucleotide excision repair. Here we describe the construction of three different XPC-/-/CSB-/- human cell lines that, contrary to expectations, perform TCR. The XPC and CSB genes were mutated in cell lines derived from Xeroderma Pigmentosum patients as well as from normal human fibroblasts and repair was analyzed at the whole genome level using the very sensitive XR-seq method. As predicted, XPC-/- cells exhibited only TCR and CSB-/- cells exhibited only global repair. However, the XPC-/-/CSB-/- double mutant cell lines, although having greatly reduced repair, exhibited TCR. Mutating the CSA gene to generate a triple mutant XPC-/-/CSB-/-/CSA-/- cell line eliminated all residual TCR activity. Together, these findings provide new insights into the mechanistic features of mammalian nucleotide excision repair.
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Affiliation(s)
- Laura A Lindsey-Boltz
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Yanyan Yang
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Cansu Kose
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Nazli Deger
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Khagani Eynullazada
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Hiroaki Kawara
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Aziz Sancar
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
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12
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Blessing C, Apelt K, van den Heuvel D, Gonzalez-Leal C, Rother MB, van der Woude M, González-Prieto R, Yifrach A, Parnas A, Shah RG, Kuo TT, Boer DEC, Cai J, Kragten A, Kim HS, Schärer OD, Vertegaal ACO, Shah GM, Adar S, Lans H, van Attikum H, Ladurner AG, Luijsterburg MS. XPC-PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair. Nat Commun 2022; 13:4762. [PMID: 35963869 PMCID: PMC9376112 DOI: 10.1038/s41467-022-31820-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2021] [Accepted: 06/29/2022] [Indexed: 11/24/2022] Open
Abstract
Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR. Cells employ global genome nucleotide excision repair to repair a broad spectrum of genomic DNA lesions. Here, the authors reveal how chromatin is primed for repair, providing insight into mechanisms of chromatin plasticity during DNA repair.
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Affiliation(s)
- Charlotte Blessing
- Biomedical Center (BMC), Physiological Chemistry, Faculty of Medicine, LMU Munich, Planegg-Martinsried, Germany.,International Max Planck Research School (IMPRS) for Molecular Life Sciences, Planegg-Martinsried, Germany
| | - Katja Apelt
- Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands
| | - Diana van den Heuvel
- Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands
| | - Claudia Gonzalez-Leal
- Biomedical Center (BMC), Physiological Chemistry, Faculty of Medicine, LMU Munich, Planegg-Martinsried, Germany.,International Max Planck Research School (IMPRS) for Molecular Life Sciences, Planegg-Martinsried, Germany
| | - Magdalena B Rother
- Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands
| | - Melanie van der Woude
- Department of Molecular Genetics, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Román González-Prieto
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.,Genome Proteomics Laboratory, Andalusian Center For Molecular Biology and Regenerative Medicine (CABIMER), University of Seville, Seville, Spain.,Department of Cell Biology, University of Seville, Seville, Spain
| | - Adi Yifrach
- Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Avital Parnas
- Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Rashmi G Shah
- Laboratory for Skin Cancer Research, CHU-Q: Laval University Hospital Research Centre of Quebec (CHUL site), Quebec City, Canada
| | - Tia Tyrsett Kuo
- Biomedical Center (BMC), Physiological Chemistry, Faculty of Medicine, LMU Munich, Planegg-Martinsried, Germany.,International Max Planck Research School (IMPRS) for Molecular Life Sciences, Planegg-Martinsried, Germany
| | - Daphne E C Boer
- Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands
| | - Jin Cai
- Biomedical Center (BMC), Physiological Chemistry, Faculty of Medicine, LMU Munich, Planegg-Martinsried, Germany.,International Max Planck Research School (IMPRS) for Molecular Life Sciences, Planegg-Martinsried, Germany
| | - Angela Kragten
- Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands
| | - Hyun-Suk Kim
- Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea
| | - Orlando D Schärer
- Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea.,Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea
| | - Alfred C O Vertegaal
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, The Netherlands
| | - Girish M Shah
- Laboratory for Skin Cancer Research, CHU-Q: Laval University Hospital Research Centre of Quebec (CHUL site), Quebec City, Canada
| | - Sheera Adar
- Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Hannes Lans
- Department of Molecular Genetics, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Haico van Attikum
- Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands
| | - Andreas G Ladurner
- Biomedical Center (BMC), Physiological Chemistry, Faculty of Medicine, LMU Munich, Planegg-Martinsried, Germany. .,International Max Planck Research School (IMPRS) for Molecular Life Sciences, Planegg-Martinsried, Germany. .,Eisbach Bio GmbH, Planegg-Martinsried, Germany.
| | - Martijn S Luijsterburg
- Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
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13
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Shao X, Yang X, Liu Y, Song Q, Pan X, Chen W, Jiang W, Xu D, Song Y, Chen R. Genetic polymorphisms in DNA repair genes and their association with risk of cervical cancer: A systematic review and meta-analysis. J Obstet Gynaecol Res 2022; 48:2405-2418. [PMID: 35732591 DOI: 10.1111/jog.15325] [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: 06/21/2021] [Revised: 03/05/2022] [Accepted: 05/29/2022] [Indexed: 11/29/2022]
Abstract
BACKGROUND There have been a large number of epidemiologic studies regarding the association between genetic polymorphisms in DNA repair genes and onset of cervical cancer. However, results are inconsistent. METHODS Articles published before June 2021 and regarding genetic polymorphisms in DNA repair genes and cervical cancer were searched in following databases: PubMed, Web of Science, Google Scholar, and CNKI. With at least three articles for each polymorphism, we made meta-analysis to compute multivariate odds ratios (ORs) and their 95% confidence intervals (CIs). RESULTS The present study showed significant associations between XRCC1 Arg399Gln polymorphisms and risk of cervical cancer in Asian, whereas no significant association between them were showed in Caucasian (Asian: GA vs. GG: OR = 1.27, 95%CI 1.06-1.52; AA vs. GG: OR = 1.91, 95%CI 1.29-2.83; GA + AA vs. GG: OR = 1.36, 95%CI 1.12-1.65; AA vs. GG + GA: OR = 1.66, 95%CI 1.17-2.37; Caucasian: GA vs. GG: OR = 1.08, 95%CI 0.83-1.41; AA vs. GG: OR = 2.18, 95%CI 0.75-6.31; GA + AA vs. GG: OR = 1.23, 95%CI 0.85-1.78; AA vs. GG + GA: OR = 1.70, 95%CI 0.69-4.18). In addition, there were significant associations between ERCC2 rs13181 polymorphisms and risk of cervical cancer in Asian (AC vs AA: OR = 0.53, 95%CI 0.37-0.75, I2 = 0.0%, p value of Q test = 0.847; AC + CC vs AA: OR = 0.50, 95%CI 0.36-0.70, I2 = 0.0%, p value of Q test = 0.856). CONCLUSIONS The meta-analysis showed that there were significant associations between XRCC1 Arg399Gln and ERCC2 rs13181 polymorphisms and risk of cervical cancer.
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Affiliation(s)
- Xueting Shao
- Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.,Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Xiaole Yang
- Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Ying Liu
- Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Qingxia Song
- Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Xin Pan
- Department of Gynaecology and Obstetrics, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
| | - Wansu Chen
- Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.,Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Wei Jiang
- Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Dan Xu
- Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Yuanyuan Song
- Department of Gynaecology, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China
| | - Renshou Chen
- Institute of Traditional Chinese Medicine Literature, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
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14
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Zhang X, Yin M, Hu J. Nucleotide excision repair: a versatile and smart toolkit. Acta Biochim Biophys Sin (Shanghai) 2022; 54:807-819. [PMID: 35975604 PMCID: PMC9828404 DOI: 10.3724/abbs.2022054] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Nucleotide excision repair (NER) is a major pathway to deal with bulky adducts induced by various environmental toxins in all cellular organisms. The two sub-pathways of NER, global genome repair (GGR) and transcription-coupled repair (TCR), differ in the damage recognition modes. In this review, we describe the molecular mechanism of NER in mammalian cells, especially the details of damage recognition steps in both sub-pathways. We also introduce new sequencing methods for genome-wide mapping of NER, as well as recent advances about NER in chromatin by these methods. Finally, the roles of NER factors in repairing oxidative damages and resolving R-loops are discussed.
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Affiliation(s)
| | | | - Jinchuan Hu
- Correspondence address. Tel: +86-21-54237702; E-mail:
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15
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Li W, Jones K, Burke TJ, Hossain MA, Lariscy L. Epigenetic Regulation of Nucleotide Excision Repair. Front Cell Dev Biol 2022; 10:847051. [PMID: 35465333 PMCID: PMC9023881 DOI: 10.3389/fcell.2022.847051] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2022] [Accepted: 03/24/2022] [Indexed: 12/30/2022] Open
Abstract
Genomic DNA is constantly attacked by a plethora of DNA damaging agents both from endogenous and exogenous sources. Nucleotide excision repair (NER) is the most versatile repair pathway that recognizes and removes a wide range of bulky and/or helix-distorting DNA lesions. Even though the molecular mechanism of NER is well studied through in vitro system, the NER process inside the cell is more complicated because the genomic DNA in eukaryotes is tightly packaged into chromosomes and compacted into a nucleus. Epigenetic modifications regulate gene activity and expression without changing the DNA sequence. The dynamics of epigenetic regulation play a crucial role during the in vivo NER process. In this review, we summarize recent advances in our understanding of the epigenetic regulation of NER.
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16
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CSB-independent, XPC-dependent transcription-coupled repair in Drosophila. Proc Natl Acad Sci U S A 2022; 119:2123163119. [PMID: 35217627 PMCID: PMC8892495 DOI: 10.1073/pnas.2123163119] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/27/2022] [Indexed: 02/08/2023] Open
Abstract
Drosophila melanogaster has been extensively used as a model system to study ionizing radiation and chemical-induced mutagenesis, double-strand break repair, and recombination. However, there are only limited studies on nucleotide excision repair in this important model organism. An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, we recently found that the Drosophila S2 cell line performs TCR comparable to human cells. Here, we have extended this work to Drosophila at all its developmental stages. We find TCR takes place throughout the life cycle of the organism. Moreover, we find that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila.
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17
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D'Souza A, Blee AM, Chazin WJ. Mechanism of action of nucleotide excision repair machinery. Biochem Soc Trans 2022; 50:375-386. [PMID: 35076656 PMCID: PMC9275815 DOI: 10.1042/bst20210246] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Revised: 12/20/2021] [Accepted: 12/22/2021] [Indexed: 11/08/2023]
Abstract
Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.
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Affiliation(s)
- Areetha D'Souza
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37240-7917, U.S.A
| | - Alexandra M Blee
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37240-7917, U.S.A
| | - Walter J Chazin
- Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37240-7917, U.S.A
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18
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Apelt K, Lans H, Schärer OD, Luijsterburg MS. Nucleotide excision repair leaves a mark on chromatin: DNA damage detection in nucleosomes. Cell Mol Life Sci 2021; 78:7925-7942. [PMID: 34731255 PMCID: PMC8629891 DOI: 10.1007/s00018-021-03984-7] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 09/27/2021] [Accepted: 10/15/2021] [Indexed: 11/28/2022]
Abstract
Global genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair proteins to access DNA lesions buried in nucleosomal DNA. The DNA-damage sensors XPC and DDB2 recognize DNA lesions in nucleosomal DNA and initiate repair. The emerging view is that a tight interplay between XPC and DDB2 is regulated by post-translational modifications on the damage sensors themselves as well as on chromatin containing DNA lesions. The choreography between XPC and DDB2, their interconnection with post-translational modifications such as ubiquitylation, SUMOylation, methylation, poly(ADP-ribos)ylation, acetylation, and the functional links with chromatin remodelling activities regulate not only the initial recognition of DNA lesions in nucleosomes, but also the downstream recruitment and necessary displacement of GG-NER factors as repair progresses. In this review, we highlight how nucleotide excision repair leaves a mark on chromatin to enable DNA damage detection in nucleosomes.
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Affiliation(s)
- Katja Apelt
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | - Hannes Lans
- Department of Molecular Genetics, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Orlando D Schärer
- Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea.,Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea
| | - Martijn S Luijsterburg
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
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19
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Gaul L, Svejstrup JQ. Transcription-coupled repair and the transcriptional response to UV-Irradiation. DNA Repair (Amst) 2021; 107:103208. [PMID: 34416541 DOI: 10.1016/j.dnarep.2021.103208] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Revised: 08/06/2021] [Accepted: 08/07/2021] [Indexed: 02/07/2023]
Abstract
Lesions in genes that result in RNA polymerase II (RNAPII) stalling or arrest are particularly toxic as they are a focal point of genome instability and potently block further transcription of the affected gene. Thus, cells have evolved the transcription-coupled nucleotide excision repair (TC-NER) pathway to identify damage-stalled RNAPIIs, so that the lesion can be rapidly repaired and transcription can continue. However, despite the identification of several factors required for TC-NER, how RNAPII is remodelled, modified, removed, or whether this is even necessary for repair remains enigmatic, and theories are intensely contested. Recent studies have further detailed the cellular response to UV-induced ubiquitylation and degradation of RNAPII and its consequences for transcription and repair. These advances make it pertinent to revisit the TC-NER process in general and with specific discussion of the fate of RNAPII stalled at DNA lesions.
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Affiliation(s)
- Liam Gaul
- Department of Cellular and Molecular Medicine, Panum Institute, Blegdamsvej 3B, University of Copenhagen, 2200, Copenhagen N, Denmark
| | - Jesper Q Svejstrup
- Department of Cellular and Molecular Medicine, Panum Institute, Blegdamsvej 3B, University of Copenhagen, 2200, Copenhagen N, Denmark.
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20
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Andrade MJ, Van Lonkhuyzen DR, Upton Z, Satyamoorthy K. RPA facilitates rescue of keratinocytes from UVB radiation damage through insulin-like growth factor-I signalling. J Cell Sci 2021; 134:jcs255786. [PMID: 34137442 DOI: 10.1242/jcs.255786] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2020] [Accepted: 05/10/2021] [Indexed: 01/19/2023] Open
Abstract
UVBR-induced photolesions in genomic DNA of keratinocytes impair cellular functions and potentially determine the cell fate post-irradiation. The ability of insulin-like growth factor-I (IGF-I) to rescue epidermal keratinocytes after photodamage via apoptosis prevention and photolesion removal was recently demonstrated using in vitro two-dimensional and three-dimensional skin models. Given the limited knowledge of specific signalling cascades contributing to post-UVBR IGF-I effects, we used inhibitors to investigate the impact of blockade of various signalling mediators on IGF-I photoprotection. IGF-I treatment, in the presence of signalling inhibitors, particularly TDRL-505, which targets replication protein A (RPA), impaired activation of IGF-1R downstream signalling, diminished cyclobutane pyrimidine dimer removal, arrested growth, reduced cell survival and increased apoptosis. Further, the transient partial knockdown of RPA was found to abrogate IGF-I-mediated responses in keratinocytes, ultimately affecting photoprotection and, thereby, establishing that RPA is required for IGF-I function. Our findings thus elucidate the importance of RPA in linking the damage response activation, cell cycle regulation, repair and survival pathways, separately initiated by IGF-I upon UVBR-induced damage. This information is potentially imperative for the development of effective sunburn and photodamage repair strategies. This article has an associated First Person interview with the first author of the paper.
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Affiliation(s)
- Melisa J Andrade
- Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India
- Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland 4059, Australia
| | - Derek R Van Lonkhuyzen
- Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland 4059, Australia
| | - Zee Upton
- Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland 4059, Australia
- Skin Research Institute of Singapore, Agency for Science, Technology and Research, Singapore138648
| | - Kapaettu Satyamoorthy
- Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India
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21
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The ECCR1 rs11615, ERCC4 rs2276466, XPC rs2228000 and XPC rs2228001 polymorphisms increase the cervical cancer risk and aggressiveness in the Bangladeshi population. Heliyon 2021; 7:e05919. [PMID: 33490679 PMCID: PMC7809183 DOI: 10.1016/j.heliyon.2021.e05919] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2020] [Revised: 12/07/2020] [Accepted: 01/05/2021] [Indexed: 11/21/2022] Open
Abstract
Background Multiple studies around the world revealed that genetic polymorphism in different genes of the DNA repair system might affect the DNA repair capabilities and accelerate the chances of cervical cancer (CC) development. Therefore, we aimed to evaluate the association of DNA repair gene- ECCR1 rs11615, ERCC4 rs2276466, XPC rs2228000 and rs2228001 polymorphisms and CC susceptibility in the Bangladeshi population. Methods A case-control genetic association study was conducted among 210 patients with diagnostically confirmed CC and 200 healthy volunteers. The p-value and OR (odds ratios) with 95% CI (confidence interval) were evaluated to get the level of association. Results After the individual analysis of all SNPs, we noticed that ECCR1 rs11615 possessed a significantly lower risk, whereas ERCC4 rs2276466 possessed a significantly elevated risk of CC in all genetic models (p < 0.05). XPC rs2228000 showed a significantly lower risk of CC in TC, TC + CC genotypes and allele model (OR = 0.61, p = 0.025; OR = 0.61, p = 0.019 and OR = 0.67, p = 0.027, respectively), whereas XPC rs2228001 possessed a significantly elevated risk of CC in CA, CA + AA genotypes and allele model (OR = 1.67, p = 0.012; OR = 1.69, p = 0.009 and OR = 1.42, p = 0.022). Besides, ERCC4 rs2276466 (Grade III vs. I + II: OR = 4.01, p = 0.003) and XPC rs2228001 (Grade III vs. I + II: OR = 3.38, p = 0.003) were connected with high tumor aggressiveness and ERCC4 rs2276466 was also showed a lower risk of CC development in the younger population (<45 years). Conclusion The findings supported that rs2276466 and rs2228001 polymorphisms increase CC development and aggressiveness, whereas rs11615 and rs2228000 lower the CC risk in the studied population.
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22
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Chauhan AK, Li P, Sun Y, Wani G, Zhu Q, Wani AA. Spironolactone-induced XPB degradation requires TFIIH integrity and ubiquitin-selective segregase VCP/p97. Cell Cycle 2020; 20:81-95. [PMID: 33381997 PMCID: PMC7849777 DOI: 10.1080/15384101.2020.1860559] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Mineralocorticoid and androgen receptor antagonist, spironolactone, was recently identified as an inhibitor of nucleotide excision repair (NER), acting via induction of proteolysis of TFIIH component Xeroderma Pigmentosum B protein (XPB). This activity provides a strong rationale for repurposing spironolactone for cancer therapy. Here, we report that the spironolactone-induced XPB proteolysis is mediated through ubiquitin-selective segregase, valosin-containing protein (VCP)/p97. We show that spironolactone induces a dose- and time-dependent degradation of XPB but not XPD, and that the XPB degradation is blocked by VCP/p97 inhibitors DBeQ, NMS-873, and neddylation inhibitor MLN4924. Moreover, the cellular treatment by VCP/p97 inhibitors leads to the accumulation of ubiquitin conjugates of XPB but not XPD. VCP/p97 knockdown by inducible shRNA does not affect XPB level but compromises the spironolactone-induced XPB degradation. Also, VCP/p97 interacts with XPB upon treatment of spironolactone and proteasome inhibitor MG132, while the VCP/p97 adaptor UBXD7 binds XPB and its ubiquitin conjugates. Additionally, ATP analog-mediated inhibition of Cdk7 significantly decelerates spironolactone-induced XPB degradation. Likewise, engaging TFIIH to NER by UV irradiation slows down spironolactone-induced XPB degradation. These results indicate that the spironolactone-induced XPB proteolysis requires VCP/p97 function and that XPB within holo-TFIIH rather than core-TFIIH is more vulnerable to spironolactone-induced proteolysis. Abbreviations
NER: nucleotide excision repair; TFIIH: transcription factor II H; CAK: Cdk-activating kinase (CAK) complex; XPB: Xeroderma Pigmentosum type B; VCP/p97: valosin-containing protein/p97; Cdk7: cyclin-dependent kinase 7; NAE: NEDD8-activating enzyme; IP: immunoprecipitation
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Affiliation(s)
- Anil K Chauhan
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Ping Li
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Yingming Sun
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Gulzar Wani
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Qianzheng Zhu
- Department of Radiology, The Ohio State University , Columbus, OH, USA
| | - Altaf A Wani
- Department of Radiology, The Ohio State University , Columbus, OH, USA.,Department of Molecular and Cellular Biochemistry, The Ohio State University , Columbus, OH, USA.,James Cancer Hospital and Solove Research Institute, The Ohio State University , Columbus, OH, USA
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Chen Y, Geng A, Zhang W, Qian Z, Wan X, Jiang Y, Mao Z. Fight to the bitter end: DNA repair and aging. Ageing Res Rev 2020; 64:101154. [PMID: 32977059 DOI: 10.1016/j.arr.2020.101154] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Revised: 08/05/2020] [Accepted: 08/19/2020] [Indexed: 12/11/2022]
Abstract
DNA carries the genetic information that directs complex biological processes; thus, maintaining a stable genome is critical for individual growth and development and for human health. DNA repair is a fundamental and conserved mechanism responsible for mending damaged DNA and restoring genomic stability, while its deficiency is closely related to multiple human disorders. In recent years, remarkable progress has been made in the field of DNA repair and aging. Here, we will extensively discuss the relationship among DNA damage, DNA repair, aging and aging-associated diseases based on the latest research. In addition, the possible role of DNA repair in several potential rejuvenation strategies will be discussed. Finally, we will also review the emerging methods that may facilitate future research on DNA repair.
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24
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Hakim MS, Annisa L, Jariah ROA, Vink C. The mechanisms underlying antigenic variation and maintenance of genomic integrity in Mycoplasma pneumoniae and Mycoplasma genitalium. Arch Microbiol 2020; 203:413-429. [PMID: 32970220 DOI: 10.1007/s00203-020-02041-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Revised: 08/02/2020] [Accepted: 09/12/2020] [Indexed: 11/28/2022]
Abstract
Mycoplasma pneumoniae and Mycoplasma genitalium are important causative agents of infections in humans. Like all other mycoplasmas, these species possess genomes that are significantly smaller than that of other prokaryotes. Moreover, both organisms possess an exceptionally compact set of DNA recombination and repair-associated genes. These genes, however, are sufficient to generate antigenic variation by means of homologous recombination between specific repetitive genomic elements. At the same time, these mycoplasmas have likely evolved strategies to maintain the stability and integrity of their 'minimal' genomes. Previous studies have indicated that there are considerable differences between mycoplasmas and other bacteria in the composition of their DNA recombination and repair machinery. However, the complete repertoire of activities executed by the putative recombination and repair enzymes encoded by Mycoplasma species is not yet fully understood. In this paper, we review the current knowledge on the proteins that likely form part of the DNA repair and recombination pathways of two of the most clinically relevant Mycoplasma species, M. pneumoniae and M. genitalium. The characterization of these proteins will help to define the minimal enzymatic requirements for creating bacterial genetic diversity (antigenic variation) on the one hand, while maintaining genomic integrity on the other.
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Affiliation(s)
- Mohamad S Hakim
- Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, 55281, Yogyakarta, Indonesia. .,Postgraduate School of Molecular Medicine, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands.
| | - Luthvia Annisa
- Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, 55281, Yogyakarta, Indonesia
| | - Rizka O A Jariah
- Department of Health Science, Faculty of Vocational Studies, Universitas Airlangga, Surabaya, Indonesia
| | - Cornelis Vink
- Department of Life Sciences, Erasmus University College, Erasmus University, 3011 HP, Rotterdam, The Netherlands.
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25
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Protection from Ultraviolet Damage and Photocarcinogenesis by Vitamin D Compounds. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1268:227-253. [PMID: 32918222 DOI: 10.1007/978-3-030-46227-7_12] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Exposure of skin cells to UV radiation results in DNA damage, which if inadequately repaired, may cause mutations. UV-induced DNA damage and reactive oxygen and nitrogen species also cause local and systemic suppression of the adaptive immune system. Together, these changes underpin the development of skin tumours. The hormone derived from vitamin D, calcitriol (1,25-dihydroxyvitamin D3) and other related compounds, working via the vitamin D receptor and at least in part through endoplasmic reticulum protein 57 (ERp57), reduce cyclobutane pyrimidine dimers and oxidative DNA damage in keratinocytes and other skin cell types after UV. Calcitriol and related compounds enhance DNA repair in keratinocytes, in part through decreased reactive oxygen species, increased p53 expression and/or activation, increased repair proteins and increased energy availability in the cell when calcitriol is present after UV exposure. There is mitochondrial damage in keratinocytes after UV. In the presence of calcitriol, but not vehicle, glycolysis is increased after UV, along with increased energy-conserving autophagy and changes consistent with enhanced mitophagy. Reduced DNA damage and reduced ROS/RNS should help reduce UV-induced immune suppression. Reduced UV immune suppression is observed after topical treatment with calcitriol and related compounds in hairless mice. These protective effects of calcitriol and related compounds presumably contribute to the observed reduction in skin tumour formation in mice after chronic exposure to UV followed by topical post-irradiation treatment with calcitriol and some, though not all, related compounds.
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26
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Li W, Sancar A. Methodologies for detecting environmentally induced DNA damage and repair. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2020; 61:664-679. [PMID: 32083352 PMCID: PMC7442611 DOI: 10.1002/em.22365] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/25/2019] [Revised: 02/08/2020] [Accepted: 02/16/2020] [Indexed: 05/07/2023]
Abstract
Environmental DNA damaging agents continuously challenge the integrity of the genome by introducing a variety of DNA lesions. The DNA damage caused by environmental factors will lead to mutagenesis and subsequent carcinogenesis if they are not removed efficiently by repair pathways. Methods for detection of DNA damage and repair can be applied to identify, visualize, and quantify the DNA damage formation and repair events, and they enable us to illustrate the molecular mechanisms of DNA damage formation, DNA repair pathways, mutagenesis, and carcinogenesis. Ever since the discovery of the double helical structure of DNA in 1953, a great number of methods have been developed to detect various types of DNA damage and repair. Rapid advances in sequencing technologies have facilitated the emergence of a variety of novel methods for detecting environmentally induced DNA damage and repair at the genome-wide scale during the last decade. In this review, we provide a historical overview of the development of various damage detection methods. We also highlight the current methodologies to detect DNA damage and repair, especially some next generation sequencing-based methods.
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Affiliation(s)
- Wentao Li
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
| | - Aziz Sancar
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
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27
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Guardamagna I, Bassi E, Savio M, Perucca P, Cazzalini O, Prosperi E, Stivala LA. A functional in vitro cell-free system for studying DNA repair in isolated nuclei. J Cell Sci 2020; 133:jcs240010. [PMID: 32376788 DOI: 10.1242/jcs.240010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2019] [Accepted: 04/19/2020] [Indexed: 12/31/2022] Open
Abstract
Assessment of DNA repair is an important endpoint measurement when studying the biochemical mechanisms of the DNA damage response and when investigating the efficacy of chemotherapy, which often uses DNA-damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, such methods have some limitations, which are mainly due to the lack of chromatin organization in the DNA templates used. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the corresponding damage-activated cytosolic extracts, containing biotinylated dUTP, nuclei were able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 and DDB1, stimulated UVC-induced dUTP incorporation. In contrast, a DDB2PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different types of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.This article has an associated First Person interview with the first author of the paper.
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Affiliation(s)
- Isabella Guardamagna
- Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università degli Studi di Pavia, 27100 Pavia, Italy
| | - Elisabetta Bassi
- Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università degli Studi di Pavia, 27100 Pavia, Italy
| | - Monica Savio
- Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università degli Studi di Pavia, 27100 Pavia, Italy
| | - Paola Perucca
- Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università degli Studi di Pavia, 27100 Pavia, Italy
| | - Ornella Cazzalini
- Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università degli Studi di Pavia, 27100 Pavia, Italy
| | - Ennio Prosperi
- Istituto di Genetica Molecolare 'Luigi Luca Cavalli-Sforza', CNR, 27100 Pavia, Italy
| | - Lucia A Stivala
- Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università degli Studi di Pavia, 27100 Pavia, Italy
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28
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Gsell C, Richly H, Coin F, Naegeli H. A chromatin scaffold for DNA damage recognition: how histone methyltransferases prime nucleosomes for repair of ultraviolet light-induced lesions. Nucleic Acids Res 2020; 48:1652-1668. [PMID: 31930303 PMCID: PMC7038933 DOI: 10.1093/nar/gkz1229] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Revised: 12/18/2019] [Accepted: 12/23/2019] [Indexed: 02/07/2023] Open
Abstract
The excision of mutagenic DNA adducts by the nucleotide excision repair (NER) pathway is essential for genome stability, which is key to avoiding genetic diseases, premature aging, cancer and neurologic disorders. Due to the need to process an extraordinarily high damage density embedded in the nucleosome landscape of chromatin, NER activity provides a unique functional caliper to understand how histone modifiers modulate DNA damage responses. At least three distinct lysine methyltransferases (KMTs) targeting histones have been shown to facilitate the detection of ultraviolet (UV) light-induced DNA lesions in the difficult to access DNA wrapped around histones in nucleosomes. By methylating core histones, these KMTs generate docking sites for DNA damage recognition factors before the chromatin structure is ultimately relaxed and the offending lesions are effectively excised. In view of their function in priming nucleosomes for DNA repair, mutations of genes coding for these KMTs are expected to cause the accumulation of DNA damage promoting cancer and other chronic diseases. Research on the question of how KMTs modulate DNA repair might pave the way to the development of pharmacologic agents for novel therapeutic strategies.
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Affiliation(s)
- Corina Gsell
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| | - Holger Richly
- Boehringer Ingelheim Pharma, Department of Molecular Biology, Birkendorfer Str. 65, 88397 Biberach an der Riß, Germany
| | - Frédéric Coin
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, Equipe Labélisée Ligue contre le Cancer, Illkirch Cedex, Strasbourg, France
| | - Hanspeter Naegeli
- Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Winterthurerstrasse 260, 8057 Zurich, Switzerland
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29
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Chien LC, Wu YH, Ho TN, Huang YY, Hsu T. Heat stress modulates nucleotide excision repair capacity in zebrafish (Danio rerio) early and mid-early embryos via distinct mechanisms. CHEMOSPHERE 2020; 238:124653. [PMID: 31473528 DOI: 10.1016/j.chemosphere.2019.124653] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Revised: 08/21/2019] [Accepted: 08/22/2019] [Indexed: 05/20/2023]
Abstract
Discharge of heated effluent at 8-12 °C above ambient into water areas is known to retard the growth of aquatic organisms due to heat stress. Nucleotide excision repair (NER) maintains genome integrity by removing helix-distorting adducts such as UV-induced DNA lesions. This study explored how NER in zebrafish (Danio rerio) embryos at different hours post fertilization (hpf) responded to + 8.5 °C heat shock for 30 min. Our transcription-based repair assay monitoring the ability of zebrafish extracts to upregulate a UV-suppressed gene expression detected a 2-fold increase of NER capacity in 10 hpf early embryos after heat stress. In contrast, heat stress caused a mild inhibition of NER capacity in 24 hpf mid-early embryos. Heat-treated and untreated 10 hpf zebrafish extracts displayed similar levels of UV-damaged-DNA binding activities, while an apparently weaker (6-4) photoproduct (6-4 PP) binding activity was present in heat-stressed 24 hpf zebrafish extracts. Heat stress enhanced UV-induced NER in 10 hpf embryos by increasing the efficiency of damage incision/excision based on both genomic DNA electrophoresis and terminal deoxytransferase (TdT)-mediated end labeling assay. UV-irradiated embryos preexposed to heat stress produced a significantly larger amount of NER-associated DNA fragments about 20-30 nucleotides in length than embryos only heat-treated or irradiated. Correlated with its inhibitory effect on 6-4 PP damage recognition, heat stress downregulated damage incision/excision activities in 24 hpf embryos. Hence, thermal stress may positively or negatively modulate NER capacity in zebrafish embryos at different stages by targeting at the step of DNA incision/excision or damage recognition.
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Affiliation(s)
- Liu-Chun Chien
- Department of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, ROC
| | - Yu-Hsuan Wu
- Department of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, ROC
| | - Tsung-Nan Ho
- Department of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, ROC
| | - Ya-Yun Huang
- Department of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, ROC
| | - Todd Hsu
- Department of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, ROC.
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30
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Kathuria P, Singh P, Sharma P, Manderville RA, Wetmore SD. Molecular Dynamics Study of One-Base Deletion Duplexes Containing the Major DNA Adduct Formed by Ochratoxin A: Effects of Sequence Context and Adduct Ionization State on Lesion Site Structure and Mutagenicity. J Phys Chem B 2019; 123:6980-6989. [PMID: 31311268 DOI: 10.1021/acs.jpcb.9b06489] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Ochratoxin A (OTA) is a ubiquitous food toxin associated with chronic nephropathy in humans and renal carcinogenicity in rodents. The mutational spectra of cells exposed to OTA reveal that one-base deletions comprise the largest percentage (73%) of the total mutations that occur upon OTA exposure. To contribute toward understanding the prevalence of OTA-induced one-base deletion mutations, the present work uses molecular dynamics (MD) simulations to analyze the conformational preferences of one-base deletion duplexes containing OT-G, the major OTA adduct (addition product) at the C8-site of guanine. Specifically, the influence of OT-G in four possible ionization states and three sequence contexts (G1, G2 and G3 in the NarI (5'-G1G2CG3CC-3'), a prokaryotic mutational hotspot sequence) on the structure of the adducted DNA is investigated. Our data reveal that the damaged helices are stable in two (B-type (B) and stacked (S)) conformations that are structurally similar to those adopted by common N-linked C8-guanine lesions. However, the adduct ionization state and sequence context affect the degree of helical distortion and the B/S conformational heterogeneity, which will impact the lesion repair and replication outcomes. This finding correlates with the experimentally reported tissue-specific mutagenicity of OTA exposure. Furthermore, regardless of the adduct conformation, ionization state, or sequence context, more stable lesion-site interactions and lack of disruption of the flanking base pairs in the one-base deletion duplexes compared to the corresponding two-base deletion helices rationalize the greater abundance of OTA induced one-base deletions. Overall, our work provides valuable structural insights that help explain the experimentally observed mutagenicity associated with OTA.
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Affiliation(s)
- Preetleen Kathuria
- Computational Biochemistry Laboratory, Department of Chemistry and Centre for Advanced Studies in Chemistry , Panjab University , Chandigarh 160014 , India
| | - Prebhleen Singh
- Computational Biochemistry Laboratory, Department of Chemistry and Centre for Advanced Studies in Chemistry , Panjab University , Chandigarh 160014 , India
| | - Purshotam Sharma
- Computational Biochemistry Laboratory, Department of Chemistry and Centre for Advanced Studies in Chemistry , Panjab University , Chandigarh 160014 , India
| | - Richard A Manderville
- Departments of Chemistry and Toxicology , University of Guelph , Guelph , Ontario N1G 2W1 , Canada
| | - Stacey D Wetmore
- Department of Chemistry and Biochemistry , University of Lethbridge , Lethbridge , Alberta T1K 3M4 , Canada
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31
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Abstract
The nucleotide excision repair (NER) system removes a variety of types of helix-distorting lesions from DNA through a dual incision mechanism, in which the damaged nucleotide bases are excised in the form of a small, excised, damage-containing single-stranded DNA oligonucleotide (sedDNA). Damage removal leaves a gap in the DNA template that must then be filled in by the action of a DNA polymerase and ligated to the downstream phosphodiester backbone in the DNA to complete the repair reaction. Defects in damage removal, sedDNA processing, or gap filling have the potential to be mutagenic and lethal to cells, and thus several human pathologies, including cancer and aging, are associated with defects in NER. This review summarizes our current understanding of NER with a focus on the enzymes that excise sedDNAs and restore the duplex DNA to its native state in human cells.
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Affiliation(s)
- Michael G Kemp
- Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, OH, United States.
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32
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DNA Damage Focus Formation Assay. Methods Mol Biol 2019. [PMID: 31267421 DOI: 10.1007/978-1-4939-9432-8_8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
Advanced techniques allow investigating cellular DNA damage measurements. Ionizing radiation produces multiple DNA damages. Among them, DNA double strand breaks are most toxic to cells. DSBs can form mutations, chromosome aberrations, and cell killing. Although DSBs in cells can be detected directly by neutral elution, pulse field gel electrophoresis, and premature chromosome condensation, recent technologies like cellular immunocytochemistry-based fluorescence detection allow us to visualize the DSBs in cells. Here, we describe gamma-H2AX and Rad51 focus formation assay, which play an important role in DNA damage responses.
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33
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Li S, Lu H, Wang Z, Hu Q, Wang H, Xiang R, Chiba T, Wu X. ERCC1/XPF Is Important for Repair of DNA Double-Strand Breaks Containing Secondary Structures. iScience 2019; 16:63-78. [PMID: 31153042 PMCID: PMC6543133 DOI: 10.1016/j.isci.2019.05.017] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2019] [Revised: 04/02/2019] [Accepted: 05/10/2019] [Indexed: 12/15/2022] Open
Abstract
The structure-specific endonuclease ERCC1/XPF plays an important role in nucleotide excision repair and interstrand cross-link repair. In this study, we identified new functions of ERCC1/XPF in DNA double-strand break (DSB) repair. We found that the conserved function of ERCC1/XPF to remove non-homologous sequences at DSBs is a rate-limiting step for homologous recombination in mammalian cells, and more importantly, we uncovered an indispensable role of ERCC1/XPF in repair of DSBs containing DNA secondary structures, including the structure-prone AT-rich DNA sequences derived from common fragile sites and G-quadruplexes (G4s). We also demonstrated a synthetic lethal interaction of XPF with DNA translocase FANCM that is involved in removing DNA secondary structures. Furthermore, inactivation of XPF sensitizes FANCM-deficient cells to G4-interacting compounds. These results suggest an important function of ERCC1/XPF in protecting DNA secondary structures and provide a rationale for targeted treatment of FANCM-deficient tumors through inhibition of XPF.
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Affiliation(s)
- Shibo Li
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Hongyan Lu
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA; School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Zi Wang
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA; Biomedical Gerontology Laboratory, Department of Health Science and Social Welfare, School of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa 359-1192, Japan
| | - Qing Hu
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Hongjun Wang
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Rong Xiang
- School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China
| | - Takuya Chiba
- Biomedical Gerontology Laboratory, Department of Health Science and Social Welfare, School of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa 359-1192, Japan
| | - Xiaohua Wu
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
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34
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Ueda M, Matsuura K, Kawai H, Wakasugi M, Matsunaga T. Spironolactone-induced XPB degradation depends on CDK7 kinase and SCF FBXL18 E3 ligase. Genes Cells 2019; 24:284-296. [PMID: 30762924 DOI: 10.1111/gtc.12674] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2018] [Revised: 01/30/2019] [Accepted: 02/09/2019] [Indexed: 12/27/2022]
Abstract
The multisubunit complex transcription factor IIH (TFIIH) has dual functions in transcriptional initiation and nucleotide excision repair (NER). TFIIH is comprised of two subcomplexes, the core subcomplex (seven subunits) including XPB and XPD helicases and the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex (three subunits) containing CDK7 kinase. Recently, it has been reported that spironolactone, an anti-aldosterone drug, inhibits cellular NER by inducing proteasomal degradation of XPB and potentiates the cytotoxicity of platinum-based drugs in cancer cells, suggesting possible drug repositioning. In this study, we have tried to uncover the mechanism underlying the chemical-induced XPB destabilization. Based on siRNA library screening and subsequent analyses, we identified SCFFBXL18 E3 ligase consisting of Skp1, Cul1, F-box protein FBXL18 and Rbx1 responsible for spironolactone-induced XPB polyubiquitination and degradation. In addition, we showed that CDK7 kinase activity is required for this process. Finally, we found that the Ser90 residue of XPB is essential for the chemical-induced destabilization. These results led us to propose a model that spironolactone may trigger the phosphorylation of XPB at Ser90 by CDK7, which promotes the recognition and polyubiquitination of XPB by SCFFBXL18 for proteasomal degradation.
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Affiliation(s)
- Masanobu Ueda
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Kenkyo Matsuura
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Hidehiko Kawai
- Department of Experimental Oncology, Research Center for Radiation Genome Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Mitsuo Wakasugi
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Tsukasa Matsunaga
- Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
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35
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Li W, Liu W, Kakoki A, Wang R, Adebali O, Jiang Y, Sancar A. Nucleotide excision repair capacity increases during differentiation of human embryonic carcinoma cells into neurons and muscle cells. J Biol Chem 2019; 294:5914-5922. [PMID: 30808711 DOI: 10.1074/jbc.ra119.007861] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Revised: 02/22/2019] [Indexed: 11/06/2022] Open
Abstract
Embryonic stem cells can self-renew and differentiate, holding great promise for regenerative medicine. They also employ multiple mechanisms to preserve the integrity of their genomes. Nucleotide excision repair, a versatile repair mechanism, removes bulky DNA adducts from the genome. However, the dynamics of the capacity of nucleotide excision repair during stem cell differentiation remain unclear. Here, using immunoslot blot assay, we measured repair rates of UV-induced DNA damage during differentiation of human embryonic carcinoma (NTERA-2) cells into neurons and muscle cells. Our results revealed that the capacity of nucleotide excision repair increases as cell differentiation progresses. We also found that inhibition of the apoptotic signaling pathway has no effect on nucleotide excision repair capacity. Furthermore, RNA-Seq-based transcriptomic analysis indicated that expression levels of four core repair factors, xeroderma pigmentosum (XP) complementation group A (XPA), XPC, XPG, and XPF-ERCC1, are progressively up-regulated during differentiation, but not those of replication protein A (RPA) and transcription factor IIH (TFIIH). Together, our findings reveal that increase of nucleotide excision repair capacity accompanies cell differentiation, supported by the up-regulated transcription of genes encoding DNA repair enzymes during differentiation of two distinct cell lineages.
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Affiliation(s)
- Wentao Li
- From the Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Wenjie Liu
- From the Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102 China
| | - Ayano Kakoki
- From the Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Rujin Wang
- Department of Biostatistics, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Ogun Adebali
- Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul 34956 Turkey
| | - Yuchao Jiang
- Department of Biostatistics, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Aziz Sancar
- From the Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
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36
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Sugasawa K. Mechanism and regulation of DNA damage recognition in mammalian nucleotide excision repair. DNA Repair (Amst) 2019; 45:99-138. [DOI: 10.1016/bs.enz.2019.06.004] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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37
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Mu H, Geacintov NE, Broyde S, Yeo JE, Schärer OD. Molecular basis for damage recognition and verification by XPC-RAD23B and TFIIH in nucleotide excision repair. DNA Repair (Amst) 2018; 71:33-42. [PMID: 30174301 DOI: 10.1016/j.dnarep.2018.08.005] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Global genome nucleotide excision repair (GG-NER) is the main pathway for the removal of bulky lesions from DNA and is characterized by an extraordinarily wide substrate specificity. Remarkably, the efficiency of lesion removal varies dramatically and certain lesions escape repair altogether and are therefore associated with high levels of mutagenicity. Central to the multistep mechanism of damage recognition in NER is the sensing of lesion-induced thermodynamic and structural alterations of DNA by the XPC-RAD23B protein and the verification of the damage by the transcription/repair factor TFIIH. Additional factors contribute to the process: UV-DDB, for the recognition of certain UV-induced lesions in particular in the context of chromatin, while the XPA protein is believed to have a role in damage verification and NER complex assembly. Here we consider the molecular mechanisms that determine repair efficiency in GG-NER based on recent structural, computational, biochemical, cellular and single molecule studies of XPC-RAD23B and its yeast ortholog Rad4. We discuss how the actions of XPC-RAD23B are integrated with those of other NER proteins and, based on recent high-resolution structures of TFIIH, present a structural model of how XPC-RAD23B and TFIIH cooperate in damage recognition and verification.
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Affiliation(s)
- Hong Mu
- Department of Biology, New York University, New York, NY 10003, USA
| | | | - Suse Broyde
- Department of Biology, New York University, New York, NY 10003, USA
| | - Jung-Eun Yeo
- Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea
| | - Orlando D Schärer
- Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea; Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.
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38
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Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells. Sci Rep 2018; 8:2265. [PMID: 29396432 PMCID: PMC5797224 DOI: 10.1038/s41598-018-20527-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2017] [Accepted: 01/18/2018] [Indexed: 01/27/2023] Open
Abstract
Novel in vivo excision assays for monitoring the excised oligonucleotide products of nucleotide excision repair in UV-irradiated cells have provided unprecedented views of the kinetics and genomic distribution of repair events. However, an unresolved issue is the fate of the excised oligonucleotide products of repair and their mechanism of degradation. Based on our observation that decreases in excised oligonucleotide abundance coincide with the induction of apoptotic signaling in UV-irradiated cells, we considered the possibility that caspase-mediated apoptotic signaling contributes to excised oligonucleotide degradation or to a general inhibition of the excision repair system. However, genetic and pharmacological approaches to inhibit apoptotic signaling demonstrated that caspase-mediated apoptotic signaling does not affect excision repair or excised oligonucleotide stability. Nonetheless, our assay for detecting soluble DNAs produced by repair also revealed the production of larger DNAs following DNA damage induction that was dependent on caspase activation. We therefore further exploited the versatility of this assay by showing that soluble DNAs produced by both nucleotide excision repair and apoptotic signaling can be monitored simultaneously with a diverse set of DNA damaging agents. Thus, our in vivo excision repair assay provides a sensitive measure of both repair kinetics and apoptotic signaling in genotoxin-treated cells.
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39
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Chang Y, Lee WY, Lin YJ, Hsu T. Mercury (II) impairs nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos by targeting primarily at the stage of DNA incision. AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2017; 192:97-104. [PMID: 28942072 DOI: 10.1016/j.aquatox.2017.09.001] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/27/2017] [Revised: 08/30/2017] [Accepted: 09/02/2017] [Indexed: 05/20/2023]
Abstract
Mercuric ion (Hg2+) is the most prevalent form of inorganic Hg found in polluted aquatic environment. As inhibition of DNA damage repair has been proposed as one of the mechanisms of Hg2+-induced genotoxicity in aquatic animals and mammalian cells, this study explored the susceptibility of different stages of nucleotide excision repair (NER) in zebrafish (Danio rerio) embryos to Hg2+ using UV-damaged DNA as the repair substrate. Exposure of embryos at 1h post fertilization (hpf) to HgCl2 at 0.1-2.5μM for 9h caused a concentration-dependent inhibition of NER capacity monitored by a transcription-based DNA repair assay. The extracts of embryos exposed to 2.5μM Hg2+ almost failed to up-regulate UV-suppressed marker cDNA transcription. No inhibition of ATP production was observed in all Hg2+-exposed embryos. Hg2+ exposure imposed either weak inhibitory or stimulating effects on the gene expression of NER factors, while band shift assay showed the inhibition of photolesion binding activities to about 40% of control in embryos treated with 1-2.5μM HgCl2. The damage incision stage of NER in zebrafish embryos was found to be more sensitive to Hg2+ than photolesion binding capacity due to the complete loss of damage incision activity in the extracts of embryos exposed to 1-2.5μM Hg2+. NER-related DNA incision was induced in UV-irradiated embryos based on the production of short DNA fragments matching the sizes of excision products generated by eukaryotic NER. Pre-exposure of embryos to Hg2+ at 0.1-2.5μM all suppressed DNA incision/excision in UV-irradiated embryos, reflecting a high sensitivity of DNA damage incision/excision to Hg2+. Our results showed the potential of Hg2+ at environmental relevant levels to disturb NER in zebrafish embryos by targeting primarily at the stage of DNA incision/excision.
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Affiliation(s)
- Yung Chang
- Institute of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, Republic of China
| | - Wei-Yuan Lee
- Institute of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, Republic of China
| | - Yu-Jie Lin
- Institute of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, Republic of China
| | - Todd Hsu
- Institute of Bioscience and Biotechnology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan, Republic of China.
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40
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Han C, Zhao R, Kroger J, He J, Wani G, Wang QE, Wani AA. UV radiation-induced SUMOylation of DDB2 regulates nucleotide excision repair. Carcinogenesis 2017; 38:976-985. [PMID: 28981631 DOI: 10.1093/carcin/bgx076] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Accepted: 07/19/2017] [Indexed: 12/22/2022] Open
Abstract
Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.
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Affiliation(s)
| | | | | | | | | | - Qi-En Wang
- Department of Radiology.,James Cancer Hospital and Solove Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
| | - Altaf A Wani
- Department of Radiology.,James Cancer Hospital and Solove Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
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41
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Hu S, Zhang X, Chen F, Wang M. Dietary polyphenols as photoprotective agents against UV radiation. J Funct Foods 2017. [DOI: 10.1016/j.jff.2017.01.009] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
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42
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Zhong D, Cadet J. Introduction. Photochem Photobiol 2017; 93:5-6. [PMID: 28211126 DOI: 10.1111/php.12722] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2017] [Accepted: 01/18/2017] [Indexed: 01/20/2023]
Affiliation(s)
| | - Jean Cadet
- University of Sherbrooke, Sherbrooke, QC, Canada
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43
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Lindsey-Boltz LA. Bringing It All Together: Coupling Excision Repair to the DNA Damage Checkpoint. Photochem Photobiol 2016; 93:238-244. [PMID: 27861980 DOI: 10.1111/php.12667] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2016] [Accepted: 09/29/2016] [Indexed: 12/15/2022]
Abstract
Nucleotide excision repair and the ATR-mediated DNA damage checkpoint are two critical cellular responses to the genotoxic stress induced by ultraviolet (UV) light and are important for cancer prevention. In vivo genetic data indicate that these global responses are coupled. Aziz Sancar et al. developed an in vitro coupled repair-checkpoint system to analyze the basic steps of these DNA damage stress responses in a biochemically defined system. The minimum set of factors essential for repair-checkpoint coupling include damaged DNA, the excision repair factors (XPA, XPC, XPF-ERCC1, XPG, TFIIH, RPA), the 5'-3' exonuclease EXO1, and the damage checkpoint proteins ATR-ATRIP and TopBP1. This coupled repair-checkpoint system was used to demonstrate that the ~30 nucleotide single-stranded DNA (ssDNA) gap generated by nucleotide excision repair is enlarged by EXO1 and bound by RPA to generate the signal that activates ATR.
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Affiliation(s)
- Laura A Lindsey-Boltz
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC
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44
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Dakup P, Gaddameedhi S. Impact of the Circadian Clock on UV-Induced DNA Damage Response and Photocarcinogenesis. Photochem Photobiol 2016; 93:296-303. [PMID: 27861965 DOI: 10.1111/php.12662] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2016] [Accepted: 10/04/2016] [Indexed: 12/11/2022]
Abstract
The skin is in constant exposure to various external environmental stressors, including solar ultraviolet (UV) radiation. Various wavelengths of UV light are absorbed by the DNA and other molecules in the skin to cause DNA damage and induce oxidative stress. The exposure to excessive ultraviolet (UV) radiation and/or accumulation of damage over time can lead to photocarcinogenesis and photoaging. The nucleotide excision repair (NER) system is the sole mechanism for removing UV photoproduct damage from DNA, and genetic disruption of this repair pathway leads to the photosensitive disorder xeroderma pigmentosum (XP). Interestingly, recent work has shown that NER is controlled by the circadian clock, the body's natural time-keeping mechanism, through regulation of the rate-limiting repair factor xeroderma pigmentosum group A (XPA). Studies have shown reduced UV-induced skin cancer after UV exposure in the evening compared to the morning, which corresponds with times of high and low repair capacities, respectively. However, most studies of the circadian clock-NER connection have utilized murine models, and it is therefore important to translate these findings to humans to improve skin cancer prevention and chronotherapy.
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Affiliation(s)
- Panshak Dakup
- Experimental and Systems Pharmacology, College of Pharmacy, Washington State University, Spokane, WA
| | - Shobhan Gaddameedhi
- Experimental and Systems Pharmacology, College of Pharmacy, Washington State University, Spokane, WA.,Sleep and Performance Research Center, Washington State University, Spokane, WA
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45
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Kemp MG, Spandau DF, Simman R, Travers JB. Insulin-like Growth Factor 1 Receptor Signaling Is Required for Optimal ATR-CHK1 Kinase Signaling in Ultraviolet B (UVB)-irradiated Human Keratinocytes. J Biol Chem 2016; 292:1231-1239. [PMID: 27979966 DOI: 10.1074/jbc.m116.765883] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2016] [Revised: 12/08/2016] [Indexed: 01/16/2023] Open
Abstract
UVB wavelengths of light induce the formation of photoproducts in DNA that are potentially mutagenic if not properly removed by the nucleotide excision repair machinery. As an additional mechanism to minimize the risk of mutagenesis, UVB-irradiated cells also activate a checkpoint signaling cascade mediated by the ATM and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) kinases to transiently suppress DNA synthesis and cell cycle progression. Given that keratinocytes in geriatric skin display reduced activation of the insulin-like growth factor 1 receptor (IGF-1R) and alterations in DNA repair rate, apoptosis, and senescence following UVB exposure, here we used cultured human keratinocytes in vitro and skin explants ex vivo to examine how IGF-1R activation status affects ATR-CHK1 kinase signaling and the inhibition of DNA replication following UVB irradiation. We find that disruption of IGF-1R signaling with small-molecule inhibitors or IGF-1 withdrawal partially abrogates both the phosphorylation and activation of CHK1 by ATR and the accompanying inhibition of chromosomal DNA synthesis in UVB-irradiated keratinocytes. A critical protein factor that mediates both ATR-CHK1 signaling and nucleotide excision repair is replication protein A, and we find that its accumulation on UVB-damaged chromatin is partially attenuated in cells with an inactive IGF-1R. These results indicate that mutagenesis and skin carcinogenesis in IGF-1-deficient geriatric skin may be caused by defects in multiple cellular responses to UVB-induced DNA damage, including through a failure to properly suppress DNA synthesis on UVB-damaged DNA templates.
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Affiliation(s)
- Michael G Kemp
- From the Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio 45435,
| | - Dan F Spandau
- the Departments of Dermatology and.,Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, and
| | - Richard Simman
- From the Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio 45435
| | - Jeffrey B Travers
- From the Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio 45435.,the Dayton Veterans Affairs Medical Center, Dayton, Ohio 45428
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46
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Zhu Q, Wani AA. Nucleotide Excision Repair: Finely Tuned Molecular Orchestra of Early Pre-incision Events. Photochem Photobiol 2016; 93:166-177. [PMID: 27696486 DOI: 10.1111/php.12647] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Accepted: 09/15/2016] [Indexed: 12/14/2022]
Abstract
Nucleotide excision repair (NER) eliminates a broad variety of helix-distorting DNA lesions that can otherwise cause genomic instability. NER comprises two distinct subpathways: global genomic NER (GG-NER) operating throughout the genome, and transcription-coupled NER (TC-NER) preferentially removing DNA lesions from transcribing DNA strands of transcriptionally active genes. Several NER factors undergo post-translational modifications, including ubiquitination, occurring swiftly and reversibly at DNA lesion sites. Accumulating evidence indicates that ubiquitination not only orchestrates the spatio-temporal recruitment of key protein factors to DNA lesion sites but also the productive assembly of NER pre-incision complex. This review will be restricted to the latest conceptual understanding of ubiquitin-mediated regulation of initial damage sensors of NER, that is DDB, XPC, RNAPII and CSB. We project hypothetical NER models in which ubiquitin-specific segregase, valosin-containing protein (VCP)/p97, plays an essential role in timely extraction of the congregated DNA damage sensors to functionally facilitate the DNA lesion elimination from the genome.
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Affiliation(s)
- Qianzheng Zhu
- Department of Radiology, The Ohio State University, Columbus, OH
| | - Altaf A Wani
- Department of Radiology, The Ohio State University, Columbus, OH.,Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH.,James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH
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47
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Jarrett SG, D'Orazio JA. Hormonal Regulation of the Repair of UV Photoproducts in Melanocytes by the Melanocortin Signaling Axis. Photochem Photobiol 2016; 93:245-258. [PMID: 27645605 DOI: 10.1111/php.12640] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Accepted: 08/31/2016] [Indexed: 12/16/2022]
Abstract
Melanoma is the deadliest form of skin cancer because of its propensity to spread beyond the primary site of disease and because it resists many forms of treatment. Incidence of melanoma has been increasing for decades. Although ultraviolet radiation (UV) has been identified as the most important environmental causative factor for melanoma development, UV-protective strategies have had limited efficacy in melanoma prevention. UV mutational burden correlates with melanoma development and tumor progression, underscoring the importance of UV in melanomagenesis. However, besides amount of UV exposure, melanocyte UV mutational load is influenced by the robustness of nucleotide excision repair, the genome maintenance pathway charged with removing UV photoproducts before they cause permanent mutations in the genome. In this review, we highlight the importance of the melanocortin hormonal signaling axis on regulating efficiency of nucleotide excision repair in melanocytes. By understanding the molecular mechanisms by which nucleotide excision repair can be increased, it may be possible to prevent many cases of melanoma by reducing UV mutational burden over time.
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Affiliation(s)
- Stuart G Jarrett
- Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY
| | - John A D'Orazio
- Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY.,Department of Toxicology and Cancer Biology, University of Kentucky College of Medicine, Lexington, KY.,Department of Physiology, University of Kentucky College of Medicine, Lexington, KY.,Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY.,Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY
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48
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Song J, Kemp MG, Choi JH. Detection of the Excised, Damage-containing Oligonucleotide Products of Nucleotide Excision Repair in Human Cells. Photochem Photobiol 2016; 93:192-198. [PMID: 27634428 PMCID: PMC5315615 DOI: 10.1111/php.12638] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2016] [Accepted: 08/10/2016] [Indexed: 11/29/2022]
Abstract
The human nucleotide excision repair system targets a wide variety of DNA adducts for removal from DNA, including photoproducts induced by UV wavelengths of sunlight. A key feature of nucleotide excision repair is its dual incision mechanism, which results in generation of a small, damage‐containing oligonucleotide approximately 24 to 32 nt in length. Detection of these excised oligonucleotides using cell‐free extracts and purified proteins with defined DNA substrates has provided a robust biochemical assay for excision repair activity in vitro. However, the relevance of a number of in vitro findings to excision repair in living cells in vivo has remained unresolved. Over the past few years, novel methods for detecting and isolating the excised oligonucleotide products of repair in vivo have therefore been developed. Here we provide a basic outline of a sensitive and versatile in vivo excision assay and discuss how the assay both confirms previous in vitro findings and offers a number of advantages over existing cell‐based DNA repair assays. Thus, the in vivo excision assay offers a powerful tool for readily monitoring the repair of DNA lesions induced by a large number of environmental carcinogens and anticancer compounds.
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Affiliation(s)
- Jimyeong Song
- Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, Korea.,Department of Bio-Analytical Science, University of Science & Technology, Daejeon, Korea
| | - Michael G Kemp
- Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, OH
| | - Jun-Hyuk Choi
- Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, Korea.,Department of Bio-Analytical Science, University of Science & Technology, Daejeon, Korea
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49
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Kemp MG, Hu J. PostExcision Events in Human Nucleotide Excision Repair. Photochem Photobiol 2016; 93:178-191. [PMID: 27645806 DOI: 10.1111/php.12641] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2016] [Accepted: 08/26/2016] [Indexed: 12/27/2022]
Abstract
The nucleotide excision repair system removes a wide variety of DNA lesions from the human genome, including photoproducts induced by ultraviolet (UV) wavelengths of sunlight. A defining feature of nucleotide excision repair is its dual incision mechanism, in which two nucleolytic incision events on the damaged strand of DNA at sites bracketing the lesion generate a damage-containing DNA oligonucleotide and a single-stranded DNA gap approximately 30 nucleotides in length. Although the early events of nucleotide excision repair, which include lesion recognition and the dual incisions, have been explored in detail and are reasonably well understood, the fate of the single-stranded DNA gaps and excised oligonucleotide products of repair have not been as extensively examined. In this review, recent findings that address these less-explored aspects of nucleotide excision repair are discussed and support the concept that postincision gap and excised oligonucleotide processing are critical steps in the cellular response to DNA damage induced by UV light and other environmental carcinogens. Defects in these latter stages of repair lead to cell death and other DNA damage signaling responses and may therefore contribute to a number of human disease states associated with exposure to UV wavelengths of sunlight, including skin cancer, aging and autoimmunity.
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Affiliation(s)
- Michael G Kemp
- Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, OH
| | - Jinchuan Hu
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC
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50
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Krasikova YS, Rechkunova NI, Lavrik OI. Replication protein A as a major eukaryotic single-stranded DNA-binding protein and its role in DNA repair. Mol Biol 2016. [DOI: 10.1134/s0026893316030080] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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