Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome |
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| Affiliation: | 1. Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil;2. CNRS-UMR8200, Université Paris Sud, Institut de Cancérologie Gustave Roussy, 94805 Villejuif, France;3. Fundación Instituto Leloir, CONICET, Buenos Aires, Argentina;4. CEA, DSV-iMETI-SEPIA, BP6, 92265 Fontenay-aux-Roses, France;1. Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan;2. Department of Physiological Sciences, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan;3. Department of Life Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan;1. Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA;2. Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, 28040 Madrid, Spain;3. Department of Biomedical Research, National Jewish Health, Denver, CO 80206, USA;1. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada;2. Structural Cell Biology Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, US Department of Health and Human Services, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA;3. Repare Therapeutics, 7210 Frederick-Banting, Suite 100, St-Laurent, QC H4S 2A1, Canada;4. Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada;5. National Research Council Canada Human Health Therapeutics Research Center, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada;6. MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK;1. Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA;1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA |
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| Abstract: | Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/PolηKD cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype. |
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| Keywords: | UV damage bypass NER TLS DNA polymerase η Cell cycle progression Replication fork DNA strand breaks γH2AX |
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