Abstract
Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart.
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Acknowledgements
We would like to thank Dr H Chun for providing purified ATM kinase. This research was supported by grants from NIH (CA92111 and CA151367 to S-HL), the IU Cancer Center and the Walther Oncology Center. BDB was supported by NIH predoctoral training grant (NIDDK 1 T32 DK 0719-15). We acknowledge K Valerie for the kind gift of the adenovirus I-SceI system, the support of NIH R01 GM084020 and NIH R01 CA100862 (J.N.), an APRC supplement CA100862 (JN and RH), NIH R01 CA102283 (RH), NIH R01 HL075783 (RH), NIH R01 CA139429 (RH), and the Leukemia and Lymphoma Society SCOR 7388-06 (RH).
Author contributions: RH conceived of and analyzed the experiments, and wrote the manuscript. SF, EAW, Y-JL, S-JP, BDB and J-S Y performed and analyzed the experiments. AL performed the modeling analysis. JAN analyzed experiments and wrote the manuscript. S-HL designed experiments, analyzed data and wrote the manuscript.
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Hromas, R., Williamson, E., Fnu, S. et al. Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart. Oncogene 31, 4245–4254 (2012). https://doi.org/10.1038/onc.2011.586
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DOI: https://doi.org/10.1038/onc.2011.586
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