Maeshima K, Maraboeuf F, Morimatsu K, Horii T, Takahashi M
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
J Mol Biol. 1998 Dec 4;284(3):689-97. doi: 10.1006/jmbi.1998.2225.
Rad51 is a eukaryotic homologue of RecA and it catalyzes the DNA strand exchange reaction in homologous recombination. This protein, like RecA, requires ATP as a cofactor for activity. We investigated the mechanism of activation of this protein by the nucleotide cofactor by studying the effect of various nucleotides, particularly ATP, ADP and the non-hydrolyzable analog of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) on the DNA binding of a Xenopus Rad51 protein (XRad51.1). DNA binding was studied in solution by monitoring the fluorescence changes of etheno-modified fluorescent poly(dA) or fluorescein-labeled oligo(dT) and by filter binding assay. Active nucleotides (ATP, dATP) changed the DNA binding mode of XRad51.1. In the active complex, the DNA bases were destacked and their motion was highly restricted. Dissociation of XRad51.1 from DNA was accelerated by ATP and dATP, as was dissociation of RecA from DNA. In contrast to these similarities with RecA, the XRad51.1-DNA complex was dissociated by the non-hydrolyzable analog of ATP (ATPgammaS) and this dissociation was not significantly accelerated by ADP. The effect of ATP hydrolysis on the XRad51.1-DNA complex differs from that on the RecA-DNA complex. ATP hydrolysis may not be essential for the strand exchange reaction whereas the changes in the DNA structure by ATP are important.
Rad51是RecA的真核同源物，它在同源重组中催化DNA链交换反应。这种蛋白质与RecA一样，需要ATP作为活性的辅助因子。我们通过研究各种核苷酸，特别是ATP、ADP和ATP的不可水解类似物腺苷 - 5'-O-(3-硫代三磷酸)(ATPγS)对非洲爪蟾Rad51蛋白(XRad51.1)DNA结合的影响，来研究核苷酸辅助因子对该蛋白的激活机制。通过监测乙烯修饰的荧光聚(dA)或荧光素标记的寡聚(dT)的荧光变化以及滤膜结合试验，在溶液中研究DNA结合。活性核苷酸(ATP、dATP)改变了XRad51.1的DNA结合模式。在活性复合物中，DNA碱基堆积被破坏，其运动受到高度限制。ATP和dATP加速了XRad51.1从DNA上的解离，RecA从DNA上的解离也是如此。与这些与RecA的相似之处相反，XRad51.1-DNA复合物被ATP的不可水解类似物(ATPγS)解离，并且这种解离不会被ADP显著加速。ATP水解对XRad51.1-DNA复合物的影响与对RecA-DNA复合物的影响不同。ATP水解可能不是链交换反应所必需的，而ATP引起的DNA结构变化是重要的。
