Blocked RecA protein-mediated DNA strand exchange reactions are reversed by the RuvA and RuvB proteins.
RecA protein is unable to complete a DNA strand exchange reaction between a circular single-stranded DNA and a linear duplex DNA substrate with heterologous sequences of 375 base pairs at the distal end. Instead, it generates a branched intermediate in which strand exchange has proceeded up to the homology/heterology junction. Addition of the RuvA and RuvB proteins to these stalled intermediates leads to the rapid conversion of intermediates back to the original substrates. The reversal reaction is initiated at the branch, and the hybrid DNA is unwound in the direction opposite to that of the RecA reaction that created it. Under optimal conditions the rate of the reaction exhibits only a modest dependence on the length of hybrid DNA that must be unwound. Products of the reversal reaction are detected within minutes after addition of RuvAB, and appear with an apparent first order progress curve, exhibiting a t1/2 in the range of 6-12 min under optimal conditions. Few molecules that have undergone only partial reversal are detected. This suggests that the assembly or activation of RuvAB on the branched substrate is rate-limiting, while any migration of RuvAB on the DNA to effect unwinding of the hybrid DNA (and reformation of substrate DNA) is very fast. The results are discussed in context of the role of RuvA and RuvB proteins in recombinational DNA repair. We suggest that one function of the RuvAB proteins is to act as an antirecombinase, to eliminate intragenomic crossovers between homologous segments of the bacterial chromosome that might otherwise lead to deleterious inversions or deletions.
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Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison 53706, USA.
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D001426:Bacterial Proteins / Q000378:metabolism*; D004265:DNA Helicases; D004279:DNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008854:Microscopy, Electron; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; D011938:Rec A Recombinases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases