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 and γ-H2AX form an acid-stable biochemical complex on chromatin after DNA damage. Maximal association of BRCA1 with γ-H2AX correlates with reduced global γ-H2AX levels on chromatin late in the repair process. Since BRCA1 is known to have E3 ubiquitin ligase activity in vitro, we examined H2AX for evidence of ubiquitination. We found that H2AX is ubiquitinated at lysines 119 and 119 in vivo and that blockage of 26S proteasome function stabilizes γ-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 with γ-H2AX and that the complex is involved in attenuation of the γ-H2AX repair signal after DNA damage.
Highlights
One of the first observable responses to DNA damage is activation of DNA-PK family kinases and resulting phosphorylation of the histone variant H2AX on S139 [1]
We show that a phosphomimetic of H2AX (H2AX-E139) is ubiquitinated in vivo, and the major site of ubiquitination is on K118 and/or K119
Before DNA damage, small amounts of BRCA1 are present in the chromatin fraction, consistent with the percentage of S-phase nuclei in this population of cells [4, 7, 26]
Summary
One of the first observable responses to DNA damage is activation of DNA-PK family kinases and resulting phosphorylation of the histone variant H2AX on S139 [1]. The tail region of H2AX includes a conserved SQ motif (S139Q140) that is recognized as the core target motif of DNA-PK family serine/threonine kinases (ATM [3], ATR [4], and DNA-PK [3, 5]). After DNA damage, several proteins are recruited to regions of γ-H2AX staining. These include the breast cancer susceptibility gene BRCA1, RAD51 [7], the NBS1/RAD50/MRE11 complex [1, 8], 53BP1 [9, 10], and MDC1 [11, 12]. ATM is the major H2AX kinase in response to γ-irradiation [3] while ATR plays a larger role during DNA synthesis [4]. S139 phosphorylation of H2AX is greatly reduced in ATM/ATR knockout cells and is completely blocked by treatment with wortmannin [3], an inhibitor of DNA-PK kinases
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