American Association for Cancer Research
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Supplementary Figures from DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair

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posted on 2023-03-31, 00:10 authored by Songmin Ying, Zhihui Chen, Annette L. Medhurst, Jessica A. Neal, Zhengqiang Bao, Oliver Mortusewicz, Joanna McGouran, Xinming Song, Huahao Shen, Freddie C. Hamdy, Benedikt M. Kessler, Katheryn Meek, Thomas Helleday

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.

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National Natural Science Foundation of China

Zhejiang Provincial Natural Science Foundation

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ARTICLE 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. Cancer Res; 76(5); 1078–88. ©2015 AACR.

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