Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart

Abstract

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. While there has been a distinguished effort in identifying effectors of Chk1 activity, there are still mechanisms of its activities that are yet to be identified. Metnase/SETMAR is a SET and transposase domain protein that promotes both DNA double strand break (DSB) repair and re-start 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 an artificially induced DSB in an engineered cell system. However, an S495A Metnase mutant, which could not be phosphorylated by Chk1, had a defect in its DSB chromatin association. The S495A mutant also failed to support repair of an induced DSB when compared with wild type (wt) Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared to wt Metnase. Thus, S495 phosphorylation of Metnase differentiates between its two main 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.

The DNA repair component Metnase regulates Chk1 stability

“…We previously found that the unphosphorylated Metnase species is far better at restarting replication forks than the phosphorylated species, implying that Chk1 activity inhibited Metnase’s fork restart capability [8]. These data here, together with that previous report, suggest a layer of complexity in Chk1 signaling not currently appreciated.…”

“…We discovered that Metnase is phosphorylated at S495 in response to DNA damage by Chk1 [8]. Phosphorylation of Metnase increased its DNA DSB repair capacity, but decreased its replication fork restart activity.…”

Biological function and regulation of histone and non-histone lysine methylation in response to DNA damage

“…The increase of H3K36me2 at I-SceI-induced DSB site is mediated by recruiting Metnase (also SETMAR) and is required for NHEJ repair [47], which is in agreement with the role of Metnase in NHEJ [77,78]. The recruitment of Metnase relies on phosphorylation of Ser495 by Chk1, but not by ATM [79]. We also found that dissociation of H3K36me2-specific demethylase JHDM1a/KDM2A with chromatin is also critical for this process, and this dissociation between KDM2A and chromatin is a result of ATM-dependent phosphorylation on Thr632 [76].…”

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme

“…Because SETMAR has been reported to act specifically at sites of DNA damage, it may be that methylation only occurs on a small fraction of the total substrate pool, perhaps to shut down splicing at sites of DNA damage (4). It could also be that SETMAR activity depends on upstream activities, such as phosphorylation by Chk1 (34).…”