Single Molecule Study Of The Mechanism Of Homology Recombination Mediated By RecA

Homologous recombination(HR)is essential for maintaining the genome fidelity and generating genetic diversity.As a prototypical member of the recombinases,Rec A from Escherichia coli has been extensively studied by using single-molecule FRET(sm FRET),magnetic tweezers,optical tweezers,etc.However,these methods cannot meet the needs of wide-ranged observations nor high spatial resolution at the same time.For sequence comparison,the average base-to-base distance of the homologous ds DNA will be stretched from 0.34 nm to 0.51 nm.The increment for per base pair is 0.17 nm,which is far beyond the spatial resolution of magnetic tweezers so that it cannot be directly measured.As a high-resolution technique,the sm FRET enables us to observe more details of reactions.However,its valid measuring distance is 3-8 nm,which limits the observation range.Previous studies base on methods mentioned above show 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.In this paper,we propose an approach by combining magnetic tweezers with DNA hairpin,which meet the needs of wide-ranged observations and high spatial resolution at the same time in the study of HR.By using this approach and sm FRET to catch transient intermediates in Rec A-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 ss DNA-Rec A 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.