Supplementary Figures. Sup. Fig. 1: Recuirtment of XRCC1 to stalled DNA replication forks is prevented by CtIP. Sup. Fig. 2: CK2 actiivty is required for stabilization of XRCC1 protein and its recuirtment to stalled forks. Sup. Fig. 3: Interaction of XRCC1 and DNA-PKcs complex is not essential for recruitment of phosphorylated DNA-PKcs foci to stalled forks. Sup. Fig. 4: DNA-PKcs is phosphorylated and recruited to sites of stalled replication forks. Sup. Fig. 5: Roles of DNA-PKcs in the recovery of replication stress-induced damage. Sup. Fig. 6: DNA-PKcs recruits XRCC1 to stalled replication forks.
ARTICLE ABSTRACTA 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. Cancer Res; 76(5); 1078–88. ©2015 AACR.