BRCA1 Forms a Functional Complex with -H2AX as a Late Response to Genotoxic Stress
Figure 1
BRCA1 moves into a chromatin complex containing S139 phosphorylated H2AX (-H2AX) after DNA damage in vivo. HBL100 cells were exposed to 10 Gy -irradiation (IR) or 4 M adriamycin then allowed to recover for the indicated time before isolation of protein complexes. (a) Nonchromatin (soluble) nuclear proteins from panel (a) were immunoprecipitated (IP) with antisera to BRCA1. Immunoprecipitates were treated with phosphatase (CIP) and immunoblotted (WB) using antibodies to RNA polymerase II (Pol II, 8WG16), FCP1, or BRCA1. 10% of the lysate was immunoblotted directly and probed for -actin. (b) Total -H2AX and BRCA1-associated -H2AX were immunoprecipitated from the chromatin fraction of cells following IR. 10% of the chromatin fraction was blotted directly and probed for total H2B. (c) BRCA1 complexes identified in (a) and (b) were quantified and graphed as a function of time. (d) Chromatin-associated protein complexes were extracted (chromatin fraction) of cells exposed to IR as indicated. BRCA1 was immunoprecipitated (IP) from 90% of the chromatin extract, and immunoblots (WB) were probed with an antibody to -H2AX (S139 phosphorylated H2AX). 10% of the chromatin fraction was blotted directly and probed for total H2A. (e) Cells were pretreated with wortmannin for 15 minutes prior to treatment with 4 M adriamycin for 1 hr. 90% of the chromatin fraction was immunoprecipitated (IP) with antibodies to either BRCA1 or -H2AX and blotted (WB) with an antibody to -H2AX. 10% of the chromatin fraction was blotted directly and probed for total H2A.
