Stepwise strand exchange during RecA-induced homologous recombination

Abstract

Homologous recombination (HR) which is important for maintaining the genetic stability of the cell is mainly mediated by recombinase. As a critical recombinase exists in prokaryotic organism, RecA has been widely studied. RecA could bind single strand DNA to form a filament structure to perform strand invasion and exchange with homologous DNA. The basic strand exchange step is a pathway to investigating the mechanism of strand exchange process. However, the specific step length is still controversial. The mainstream view is to regard the basic step as 3 bp, which sounds reasonable because the basic unit of RecA filament is a RecA monomer which binds 3 base pairs, but our previous study found that the most probable basic strand exchange step is 9 bp. In this research, we set up a series of experiments to demonstrate that the basic strand exchange step is 9 bp and how the mismatch base pairs in 9 bp affect the strand exchange process. We ues digestion protection to confirm whether the strand exchange process is completed and we employ a magnetic tweezer to measure how many base pairs are exchanged by strand exchange process. The digestion protection experiments show the strand exchange cannot overcome 33% mismatch sequence, but according to the result that strand exchange could overcome a single mismatch base pair, so that the step is not 3 bp. According to strand exchange length of 33% mismatch sequence, we find a 9 bp interval between the main peaks, which implies that the strand exchange step should be 9 bp rather than 3 bp. We also use continuous mismatch base pairs instead of periodic mismatch sequence to see whether the strand exchange process can be overcome. We find that the more the mismatch base pairs, the harder the strand exchange process passes across. Homology degree and its distribution both affect the strand exchange process. In general, the results suggest that the strand exchange step mediated by RecA is 9 bp, and the number of mismatched base pairs and their distribution influence the strand exchange process. The combination of digestion protection assay and magnetic tweezers can further take advantage of single molecule techniques to investigate mechanism of HR.