Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA

Abstract

BRCA2 is a tumor suppressor that functions in homologous recombination, a key genomic integrity pathway. BRCA2 interacts with RAD51, the central protein of recombination, which forms filaments on single-stranded DNA (ssDNA) to perform homology search and DNA strand invasion. We report the purification of full-length human BRCA2 and show that it binds to ~6 RAD51 molecules and promotes RAD51 binding to ssDNA coated by replication protein A (RPA), in a manner that is stimulated by DSS1.

Figure 1. Purification of human BRCA2 and interaction with RAD51

(a) Schematic representation of purified human BRCA2 protein. (b) BRCA2 purification analysis. Left: Silver-stained fractions from purification steps: I, lysate. II, pooled fraction after ammonium sulfate precipitation. III, pooled fraction of GSTrap eluate. IV, fraction from anti-FLAG column eluate. Right: Analysis of FLAG column load (L), flow through (FT), and fractions by PAGE and silver staining. (c) Immunoblotting with antibodies spanning the full-length tagged BRCA2 protein as indicated in a. (d) GST pull-down assay design. (e) Immunoblots and (f) quantitation of the proteins in pull-down assay. The reactions contain 4.65 nM (50 ng) or 9.30 nM (100 ng) RAD51, 0.1 nM (10 ng) or no BRCA2, and 1 mM AMP-PNP, ADP, or ATP. R.S.: Regenerating system. Error bars represent s.d. of n=3.

Figure 2. BRCA2 promotes RAD51 binding to RPA-covered gapped DNA

(a) Assay design. (b) Immunoblots of the proteins bound to immobilized gapped DNA substrates. Reactions contain 0.2 µM (nt concentration) ssDNA, 0 or 20 nM RPA, 13.3 nM RAD51 (1 RAD51 per 15 nucleotides), and 0 or 0.4 nM (25 ng) BRCA2. (c) Quantitation of DNA-bound RAD51 shown in b. (d) Quantitation of DNA-bound RAD51 in the presence of different nucleotide cofactors. BRCA2 was 0.32 nM (20 ng), all other components were as in b. Plotted is fold increase over controls lacking BRCA2. (e) Purification of human DSS1 protein. I, pooled fraction of chitin column eluate. II, pooled fraction of Mono-Q column eluate. (f) Immunoblots of the proteins bound to immobilized gapped DNA substrates. Reaction contained 0.2 µM (nt concentration) ssDNA, 20 nM RPA, 13.3 nM RAD51 (1 RAD51 per 15 nucleotides), 0 or 4 nM DSS1, and 0 or 0.08 nM (5 ng) BRCA2. (g) Quantitation of DNA-bound RAD51 shown in b. Plotted is fold increase over absence of DSS1. Error bars represent s.d. of n=3, and in one case n=2.