BRCA1 Forms a Functional Complex withγ-H2AX as a Late Response to Genotoxic Stress

Abstract

Following genotoxic stress, the histone H2AX becomes phosphorylated at serine 139 by the ATM/ATR family of kinases. The tumor suppressor BRCA1, also phosphorylated by ATM/ATR kinases, is one of several proteins that colocalize with phospho-H2AX (γ-H2AX) at sites of active DNA repair. Both the precise mechanism and the purpose of BRCA1 recruitment to sites of DNA damage are unknown. Here we show that BRCA1 andγ-H2AX form an acid-stable biochemical complex on chromatin after DNA damage. Maximal association of BRCA1 withγ-H2AX correlates with reduced globalγ-H2AX levels on chromatin late in the repair process. Since BRCA1 is known to have E3 ubiquitin ligase activityin vitro, we examined H2AX for evidence of ubiquitination. We found that H2AX is ubiquitinated at lysines 119 and 119in vivoand that blockage of 26S proteasome function stabilizesγ-H2AX levels within cells. When BRCA1 levels were reduced, ubiquitination of H2AX was also reduced, and the cells retained higher levels of phosphorylated H2AX. These results indicate that BRCA1 is recruited into stable complexes withγ-H2AX and that the complex is involved in attenuation of theγ-H2AX repair signal after DNA damage.