Role of Rad51-Mediated Interactions in Recombination

摘要

Mutations in the BRCA2 gene are linked to familial and sporadic breast cancer, yet the molecular function of BRCA2 protein remains largely obscure. BRCA2 protein physically interacts with the Rad51 recombinase, a member of the RAD52 epistasis group of proteins that mediate homologous recombination (HR), a major mechanism that repairs chromosomes damaged by ionizing radiation and genotoxic agents. Accordingly, BRCA2 deficient cell lines exhibit impaired HR and sensitivity to genotoxic agents. To help define the molecular function of human BRCA2, we have expressed and purified a polypeptide that harbors the BRC3 and BRC4 repeat and also the DNA binding domain of this tumor suppressor. The BRC3/4-DBD polypeptide interacts with hRad51 and binds DNA with a distinct preference for ssDNA. Importantly, we have demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling Rad51 to utilize replication protein A-coated ssDNA as recombination substrate. In isolation neither the BRC3-BRC4 repeats nor the DNA binding domain of BRCA2 performs these mediator functions. The biochemical system described in this study should be valuable for systematically dissecting the HR functions of BRCA2 and its associated proteins such as DSS1. Comprehending the manner in which BRCA2 modulates Rad51 activity and the functional integrity of the homologous recombination machinery could very well pave the way for devising new strategies in breast cancer diagnosis, prevention, and treatment.