title: DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair.
abstract: A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved.
authors.affiliation: Department of Respiratory and Critical Care Medicine of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, China. thomas.helleday@scilifelab.se yings@zju.edu.cn.
authors.forename: Songmin
authors.initials: S
authors.lastname: Ying
authors.affiliation: Department of Gastrointestinal and Pancreatic Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Oncology, CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom.
authors.forename: Annette L
authors.initials: AL
authors.lastname: Medhurst
authors.affiliation: Department of Microbiology & Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan. Department of Pathobiology & Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Neal
authors.affiliation: Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou, China.
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Bao
authors.affiliation: Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Oliver
authors.initials: O
authors.lastname: Mortusewicz
authors.affiliation: Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, United Kingdom.
authors.forename: Joanna
authors.initials: J
authors.lastname: McGouran
authors.affiliation: Department of Gastrointestinal and Pancreatic Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
authors.forename: Xinming
authors.initials: X
authors.lastname: Song
authors.affiliation: Department of Respiratory and Critical Care Medicine of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. State Key Laboratory for Respiratory Diseases, Guangzhou, China.
authors.forename: Huahao
authors.initials: H
authors.lastname: Shen
authors.affiliation: Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.
authors.forename: Freddie C
authors.initials: FC
authors.lastname: Hamdy
authors.affiliation: Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, United Kingdom.
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
authors.affiliation: Department of Microbiology & Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan. Department of Pathobiology & Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan.
authors.forename: Katheryn
authors.initials: K
authors.lastname: Meek
authors.affiliation: Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. thomas.helleday@scilifelab.se yings@zju.edu.cn.
authors.forename: Thomas
authors.initials: T
authors.lastname: Helleday
mesh_terms: D047390:Casein Kinase II / Q000502:physiology; D002460:Cell Line; D004260:DNA Repair; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins / Q000502:physiology*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D009687:Nuclear Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D047390:Casein Kinase II; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D000076228:MRE11 Homologue Protein