Abstract
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.
Highlights
From the Department of Biochemistry, College of AgricuZture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
The quantity of the intermediate diminished and the quantity of substrate increased substantially after RuvA and RuvB protein addition. This result indicates that the RuvA and RuvB proteins are converting intermediates to substrates and thereby reversing RecA protein-mediated DNA strand exchange
Our primary conclusion is that the RuvA and RuvB proteins reverse RecA protein-mediated strand exchange when a heterologous DNA insertion is present at the 3' end of'linear duplex DNA
Summary
Exchange reaction with homologous DNA substrates, RuvA and B presumably promote branch migration predominantly in the same direction as RecA protein does, enhancing the formation of completed products of DNA strand exchange (Whitby et al, 1993). The addition of RecG protein to a similar RecA protein-mediated DNA strand exchange reaction leads to its reversal. The same mutations in a wild type background produce dramatic increases in the sensitivity of bacterial cells to DNA damaging agents (Sharples et al, 1990). These studies directly implicate the RuvA and RuvB proteins in the process of recombinationaI DNA repair. RecA protein alone promotes efficient bypass of lesions, mismatches, and short insertions of heterologous DNA sequence (up to 50-100 bp) during strand exchange (Cox, 1994). The results suggest a role for RuvA and B as an antirecombinase system, capable of reversing blocked recombination events that are potentially deleterious to the bacterial genome
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