Abstract
The ATM- and Rad3-related (ATR) kinase is a master regulator of the DNA damage response, yet how ATR is activated toward different substrates is still poorly understood. Here, we show that ATR phosphorylates Chk1 and RPA32 through distinct mechanisms at replication-associated DNA double-stranded breaks (DSBs). In contrast to the rapid phosphorylation of Chk1, RPA32 is progressively phosphorylated by ATR at Ser33 during DSB resection prior to the phosphorylation of Ser4/Ser8 by DNA-PKcs. Surprisingly, despite its reliance on ATR and TopBP1, substantial RPA32 Ser33 phosphorylation occurs in a Rad17-independent but Nbs1-dependent manner invivo and invitro. Importantly, the role of Nbs1 in RPA32 phosphorylation can be separated from ATM activation and DSB resection, and it is dependent upon the interaction of Nbs1 with RPA. An Nbs1 mutant that is unable to bind RPA fails to support proper recovery of collapsed replication forks, suggesting that the Nbs1-mediated mode of ATR activation is important for the repair of replication-associated DSBs.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1651-1662 |
| Number of pages | 12 |
| Journal | Cell reports |
| Volume | 3 |
| Issue number | 5 |
| DOIs | |
| State | Published - May 30 2013 |
| Externally published | Yes |
Bibliographical note
Funding Information:We thank Dr. K. Cimprich for communicating previously unpublished results ( Duursma et al., 2013 ); Drs. S. Elledge, A. Nussenzweig, T. Paull, X. Wu, B. Chen, R. Baer, P. Concannon, and K. Komatsu for reagents; and members of the Zou lab for helpful discussions. P.H. was supported by a postdoctoral fellowship from the Swedish Research Council. A.M. is supported by an FRSQ fellowship. L.Z. is a Jim and Ann Orr Massachusetts General Hospital Research Scholar. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology to B.S. (KAKENHI 24800044) and H.T. (KAKENHI 23300363), and grants from the NIH (GM076388), the ACS (RSG-08-297), and the Federal Share of MGH Proton Program to L.Z.
