Li Xuan, Zhang Xiao-Ping, Solinger Jachen A, Kiianitsa Konstantin, Yu Xiong, Egelman Edward H, Heyer Wolf-Dietrich
Section of Microbiology, University of California, Davis, CA 95616-8665, USA.
Nucleic Acids Res. 2007;35(12):4124-40. doi: 10.1093/nar/gkm412. Epub 2007 Jun 12.
Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-gamma-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.
Rad51和Rad54是在同源重组过程中协同作用的关键蛋白质。Rad51与ATP和单链DNA形成一个突触前细丝，在同源性搜索和DNA链交换中发挥作用，但其ATP酶活性的确切作用仍知之甚少。Rad54是一种依赖ATP的双链DNA运动蛋白，它可以使Rad51从双链DNA（DNA链交换的产物复合物）上解离下来。对芽殖酵母蛋白质的动力学分析表明，野生型和Rad51-K191R突变蛋白在双链DNA上的部分细丝刺激了Rad54 ATP酶的催化效率，明确证明在这些条件下Rad54 ATP酶活性受到刺激。用Rad51-K191R以及在ATP、ADP或ATP-γ-S存在下形成的野生型Rad51-双链DNA细丝进行的实验表明，Rad51从双链DNA上的有效周转需要Rad51 ATP酶和Rad54 ATP酶的活性。Rad51-K191R突变蛋白的结果还揭示了其在与DNA结合方面的一个意外缺陷。一旦形成，Rad51-K191R-DNA细丝在电子显微镜检查下看起来正常，但显示出稳定性显著增加。Rad51-K191R蛋白中的这些生化缺陷可能导致体内突触前（细丝形成）和突触后（细丝解体）功能不足。
