Abstract
BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function.
Highlights
We have provided strong evidence that both the BRC repeats and the DNA binding activity in BRC3/4-DNA binding domain (DBD) are needed for functional interactions with Rad51
The activity of BRC3/4-DBD is specific for Rad51 as no restoration of homologous DNA pairing was seen with E. coli RecA
BRC3/4-DBD could efficiently nucleate Rad51 onto SSB-covered ssDNA, more so than when RPA-coated ssDNA was used. Based on these biochemical findings, we suggest that in homologous recombination reactions BRCA2 fulfills the two aforementioned functions documented for BRC3/4-DBD
Summary
BRC3/4-DBD harbors the human BRCA2 BRC repeats 3 and 4 (residues 1409 –1596) and the DNA binding domain (DBD) (residues 2477– 3194). To facilitate the purification of this BRCA2 polypeptide, an amino-terminal S tag [17] and a carboxyl-terminal His tag were attached to it. The tagged BRC3/4-DBD polypeptide was introduced into the pET32b vector (Novagen) that has been deleted for the amino-terminal His tag. The Escherichia coli GST-BRC3/4 (residues 1409 –1596) expression plasmid was a kind gift from Dr Wen-Hwa Lee [10]. Construction of the plasmid that expresses the amino-terminally His6-tagged DBD (residues 2449 –3224) will be described elsewhere.
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