Iterative homology checking and non-uniform stepping during RecA-mediated strand exchange

Recombinase-mediated homologous recombination (HR) in which strands are exchanged between two similar or identical DNA molecules is essential for maintaining genome fidelity and generating genetic diversity. It is believed that HR comprises two distinct stages: an initial alignment with stringent homology checking followed by stepwise heteroduplex expansion. If and how homology checking takes place during heteroduplex expansion, however, remains unknown. In addition, the number of base pairs (bp) involved in each step is still under debate. By using single-molecule approaches to catch transient intermediates in RecA-mediated HR with different degrees of homology, we show that (i) the expansion proceeds with step sizes of multiples of 3 bp, (ii) the step sizes follow wide distributions that are similar to that of initial alignment lengths, and (iii) each distribution can be divided into a short-scale and a long-scale part irrespective of the degree of homology. Our results suggest an iterative mechanism of strand exchange in which ssDNA-RecA filament interrogates double-stranded DNA using a short tract (6-15 bp) for quick checking and a long tract (>18 bp) for stringent sequence comparison. The present work provides novel insights into the physical and structural bases of DNA recombination.