Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide.
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.
San Filippo
Joseph
J
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Chi
Peter
P
Sehorn
Michael G
MG
Etchin
Julia
J
Krejci
Lumir
L
Sung
Patrick
P
D024682:BRCA2 Protein / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000378:metabolism
D024682:BRCA2 Protein; D017931:DNA Primers; D004277:DNA, Single-Stranded; D010446:Peptide Fragments; D051716:Replication Protein A; C495153:RAD51 protein, human; D051135:Rad51 Recombinase