Effect of sequence imperfection on base-triplet stepping in strand exchange by the Rad51/RecA family of recombinases

Abstract

Homologous recombination is one of the most important DNA metabolisms involved in double strand break repair, rescue and repair of stalled replication forks, and cross-over in meiosis. Evolutionarily conserved Rad51/RecA family of recombinases catalyze the pairing of homologous DNA strands and strand exchange reaction during homologous recombination. In our earlier study, we demonstrated that individual base triplet steps during the early stages of strand invasion using single-stranded DNA curtain assays. Extending the previous works, we here examine how base triplet stepping by RecA, Rad51, and Dmc1 is influenced by DNA sequence imperfections, such as single and multiple mismatches, abasic sites, and single nucleotide insertions. Our study does not only uncover interesting features of base triplet stepping that are conserved among these three phylogenetic lineages of the Rad51/RecA family, but also reveals lineage-specific behaviors reflecting properties that are unique to each recombinase. Our findings suggest that Dmc1 is tolerant of single mismatches, multiple mismatches, and even abasic sites, whereas RecA and Rad51 are not. Interestingly, the presence of single nucleotide insertion abolishes recognition of an adjacent base triplet by all three recombinases. On the basis of these findings, we describe models for how sequence imperfections may affect base triplet recognition by Rad51/RecA family members, and we discuss how these models and our results may relate to the different biological roles of RecA, Rad51, and Dmc1.