Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods.

Abstract

Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.