Rad51 uses one mechanism to drive DNA strand exchange in both directions.
The Rad51 protein of Saccharomyces cerevisiae, like its bacterial counterpart RecA, promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in vitro. However, the two proteins differ in the requirement for initiating joint molecules and in the polarity of branch migration. Whereas RecA initiates joint molecules from any type of ends on the dsDNA and branch migration proceeds exclusively in the 5'- to 3'-direction with respect to the single strand DNA substrate, initiation mediated by Rad51 requires a complementary 3' or 5' overhanging end of the linear dsDNA and branch migration proceeds in either direction. Here we report that the rates of Rad51-mediated branch migration in either the 5'- to 3'- or 3'- to 5'-directions are affected to the same extent by temperature and MgCl(2). Furthermore, branch migration in both directions is equally impeded by insertions of non-homologous sequences in the dsDNA, inserts of 6 base pairs or more being completely inhibitory. We have also found that the preference of strand exchange in the 5'- to 3'-direction does not change if RPA is replaced by Escherichia coli SSB or T4 gene 32 proteins, suggesting that the preference for the direction of strand exchange is intrinsic to Rad51. Based on these results, we conclude that Rad51-promoted branch migration in either direction occurs fundamentally by the same mechanism, quite probably by stabilizing successively formed heteroduplex base pair.
Namsaraev
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Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-4525, USA.
Berg
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D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D051135:Rad51 Recombinase; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014764:Viral Proteins / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D008274:Magnesium