Functional interactions between BRCA1 and the checkpoint kinase ATR duringgenotoxic stress

The BRCA1 gene encodes a tumor suppressor that is mutated in 50% of familia l breast cancers. The BRCA1 protein has been implicated in the DNA damage r esponse, as DNA damage induces the phosphorylation of BRCA1 and causes its recruitment into nuclear foci that contain DNA repair proteins. The ataxia- telangiectasia-mutated (ATM) gene product controls overall BRCA1 phosphoryl ation in response to gamma -irradiation (IR). In this study, we show that B RCA1 phosphorylation is only partially ATM dependent in response to IR and ATM independent in response to treatment with UV light, or the DNA replicat ion inhibitors hydroxyurea (HU) and aphidicolin (APH). We provide evidence that the kinase responsible for this phosphorylation is the ATM-related kin ase, ATR. ATR phosphorylates BRCA1 on six Ser/Thr residues, including Ser 1 423, in vitro. Increased expression of ATR enhanced the phosphorylation of BRCA1 on Ser 1423 following cellular exposure to HU dr UV light, whereas do xycycline-induced expression of a kinase-inactive ATR mutant protein inhibi ted HU- or UV light-induced Ser 1423 phosphorylation in GM847 fibroblasts, and partially suppressed the phosphorylation of this site in response to IR . Thus, ATR, like ATM, controls BRCA1 phosphorylation in vivo. Although ATR isolated from DNA-damaged cells does not show enhanced kinase activity in vitro, we found that ATR responds to DNA damage and replication blocks by f orming distinct nuclear foci at the sites of stalled replication forks. Fur thermore, ATR nuclear foci overlap with the nuclear foci formed by BRCA1. T he dramatic relocalization of ATR in response to DNA damage points to a pos sible mechanism for its ability to enhance the phosphorylation of substrate s in response to DNA damage. Together, these results demonstrate that ATR a nd BRCA1 are components of the same genotoxic. stress-responsive pathway, a nd that ATR directly phosphorylates BRCA1 in response to damaged DNA or sta lled DNA replication.