Analysis of different DNA mismatches on RecA-catalyzed recombination in vitro

BORGOGNO MARÍA V; MONTI MARIELA R.; MONTI MARIELA R., ; ARGARAÑA CARLOS E.

Congreso; XII PABMB; 2013

Homologous recombination plays key roles in the generation of genetic diversity and DNA repair in both prokaryotes and eukaryotes. In bacteria, the initial stages of recombination are catalyzed by RecA, which promotes the alignment of two DNA sequences and initiates strand exchange between them. Previous studies have demonstrated that RecA is able to sense mismatches generated during the alignment of partially divergent DNA sequences preventing its recombination. The aim of this work is to study the effect of different DNA mismatches, as well as its number and distribution on the efficiency of recombination catalyzed by RecA of Pseudomonas aeruginosa. The kinetics of the RecA-catalyzed strand exchange reaction was determined in vitro by fluorescence resonance energy transfer using synthetic oligonucleotides. Here, we show that the position of a single G-T mismatch influences the RecA-catalyzed strand exchange. In addition, the presence of three G-T mismatches severely reduced recombination. These effects were strongly dependent on the type of mismatch (G-T, T-T or C-T). We obtained similar results with RecA from Escherichia coli and the eukaryotic homologue, Dmc1. Thus, our results reveal that RecA is sensible to the mismatch type as well as its location and number during the search for homology, a property conserved in other recombinases from bacteria and eukaryotes.