Blaho J A, Wells R D
J Biol Chem. 1987 May 5;262(13):6082-8.
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.
使用硝酸纤维素滤膜结合试验，以四种具有明确核苷酸序列的DNA聚合物和四种重组质粒来测定RecA与左手螺旋Z-DNA的结合。观察到RecA与Z-DNA聚合物有2至7倍的优先结合。RecA与左手螺旋Z-DNA聚合物的结合需要ATP或其不可水解的类似物ATP(γS)，而ADP则抑制结合。与B型和Z型的复合物形成受聚合物长度影响；RecA与较长的DNA结合更好。RecA与含有超螺旋稳定Z-DNA的重组质粒的结合与对照载体基本相似；因此，未观察到RecA对Z型的优先结合。对玉米黑粉菌的Rec1蛋白和大肠杆菌RecA蛋白进行了比较实验。在我们的实验中，RecA和Rec1在结合左手螺旋Z-DNA聚合物以及结合含有B区和/或Z区的重组质粒方面具有相似的能力。RecA含有一种受左手螺旋Z-DNA刺激的ATP酶活性。这种活性不同于受右手螺旋B-DNA刺激的活性，因为它对pH升高不太敏感。在B-DNA/Z-DNA混合实验中ATP水解的动力学表明，Z-DNA RecA复合物的周转比B-DNA慢，这表明左手螺旋Z-DNA与RecA的结合更稳定。我们的结果与左手螺旋Z-DNA参与基因重组的假设一致。
