Left-handed Z-DNA binding by the recA protein of Escherichia coli

Abstract

recA binding to left-handed Z-DNA was measured using nitrocellulose filter binding assays with four DNA polymers with defined nucleotide sequences and four recombinant plasmids. Two to 7-fold preferential binding of recA to Z-DNA polymers was observed. Left-handed Z-DNA polymer binding by recA required ATP or its nonhydrolyzable analog, ATP(gamma S), while ADP inhibited binding. Complex formation with both B- and Z-forms was influenced by polymer length; recA bound longer DNAs better. recA binding to recombinant plasmids containing supercoil-stabilized Z-DNA was essentially similar to that found for the control vector; thus, no preferential binding of recA to the Z-form was observed. Comparative experiments with the rec1 protein of Ustilago maydis and the Escherichia coli recA protein were performed. In our hands, recA and rec1 have a similar capacity for binding left-handed Z-DNA polymers and for binding recombinant plasmids containing B- and/or Z-regions. recA contains a left-handed Z-DNA-stimulated ATPase activity. This activity differs from the right-handed B-DNA-stimulated activity since it is less sensitive to increasing pH. The kinetics of ATP hydrolysis in B-DNA/Z-DNA mixing experiments showed that the turnover of the Z-DNA recA complex was slower than for B-DNA suggesting that left-handed Z-DNA is more stably bound by recA. Our results are consistent with the postulate that left-handed Z-DNA is involved in genetic recombination.