Formation Of RecA Filament During The Mechanical Unzipping Of dsDNA To ssDNA: Competition With SSB Differentially Controls RecA Mediated SOS Response And Replication Repair

Abstract

RecA and its eukaryotic homologs Rad51 and Dmc1 carry out many DNA transactions: recombinational DNA damage repair, genome integration of incoming DNA, programmed recombination during meiosis, and stalled DNA replication forks processing, all reactions initiated forming a RecA ssDNA nucleoprotein filament. ssDNA/RecA interactions activate the “SOS” response through RecA co-protease activation, raising RecA concentration. RecA/ssDNA binding must be regulated such that it only occurs in specific situations: a feature involved in modulating specificity is competitive binding of SSB and RecA to ssDNA. We studied this competition using a magnetic tweezers assay system in which we follow the formation of RecA filaments, in the presence and absence of SSB, on a single ssDNA molecule obtained by mechanical unzipping of dsDNA. We examined various buffer conditions and the effects of several relevant nucleotides. When RecA and SSB tetramer are equimolar, SSB wins the competition and no stable RecA filament is observed; when RecA is in a 20-fold molar excess, stable RecA filament forms. At intermediate molar ratios mixed situations are observed. These results provide information on the competition dynamics between RecA and SSB at the single DNA molecule level. Our results confirm previous ensemble studies: RecA and SSB affinities for ssDNA provide an intrinsic differential control for RecA mediated DNA repair and recombination functions, independently by a RecA loading machinery. SOS response increases RecA concentration giving a molar ratio of RecA:SSB of ∼30:1 outcompeting SSB without need for loading factors. These studies show that a ssDNA/RecA filament is stable for much longer than any ssDNA/SSB complex examined. This could imply that the free energy for bound RecA is much lower than that for SSB.