Atomistic Studies Support a Detailed Model of RecA Mediated Homology Recognition and Strand Exchange

Abstract

The bacterial recombinase RecA mediates homologous recombination and recombinational DNA repair through homology search and strand exchange. During homologous recombination, RecA binds an incoming single-stranded DNA (ssDNA) in its primary binding site, site I, to form a nucleoprotein filament whose crystal structure is known. The structure of the final post-stand exchange structure with double stranded DNA (dsDNA) bound in site I is also known; however, despite enormous effort, the details of homology testing have remained uncertain, partly because of the dearth of information about the structure of DNA during homology testing. Recent work has suggested that homology testing begins with the dsDNA binding to the secondary RecA binding site, site II. With detailed examination of the crystal structure, we found that an intermediate structure must exist such that homology recognition does not simply proceed from the searching state to the final post-strand exchange state, suggesting that homology recognition is governed by transitions to and from the intermediate structure that differs significantly from its final position. In this work, we present evidence that ∼ 9-15 dsDNA base pairs can bind to site II in a metastable conformation where single complementary strand bases can flip and stably pair with corresponding sequence matched incoming strand bases. That stable pairing deepens the dsDNA binding, allowing time for base flipping and strand exchange which in turn allows more dsDNA bases to bind to site II. In the absence of pairing between the complementary and incoming strands, a series of transitions drives dsDNA unbinding, with little opportunity for additional base flipping. Such a system offers very extremely rapid rejection of almost all mismatched pairings, which allows bacterial genomes to be searched on biologically relevant timescales.