DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair

• Published in: Cancer research (Chicago, Ill.) Ill.), 2016-03, Vol.76 (5), p.1078-1088
• Main Authors: Ying, Songmin, Chen, Zhihui, Medhurst, Annette L, Neal, Jessica A, Bao, Zhengqiang, Mortusewicz, Oliver, McGouran, Joanna, Song, Xinming, Shen, Huahao, Hamdy, Freddie C, Kessler, Benedikt M, Meek, Katheryn, Helleday, Thomas
• Format: Article
• Language: English
• Subjects: Casein Kinase II - physiology; Cell Line; DNA Repair; DNA Replication; DNA-Activated Protein Kinase - physiology; DNA-Binding Proteins - physiology; Humans; Medicin och hälsovetenskap; MRE11 Homologue Protein; Nuclear Proteins - physiology; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases - physiology; X-ray Repair Cross Complementing Protein 1
• ISSN: 0008-5472; 1538-7445; 1538-7445
• DOI: 10.1158/0008-5472.CAN-15-0608

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.