Sugiyama Tomohiko, Kowalczykowski Stephen C
Section of Microbiology and Center for Genetics and Development, University of California, Davis, California 95616-8665, USA.
J Biol Chem. 2002 Aug 30;277(35):31663-72. doi: 10.1074/jbc.M203494200. Epub 2002 Jun 19.
The Rad51 nucleoprotein filament mediates DNA strand exchange, a key step of homologous recombination. This activity is stimulated by replication protein A (RPA), but only when RPA is introduced after Rad51 nucleoprotein filament formation. In contrast, RPA inhibits Rad51 nucleoprotein complex formation by prior binding to single-stranded DNA (ssDNA), but Rad52 protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 protein accelerates RPA displacement by Rad51 protein. This acceleration probably involves direct interactions with both Rad51 protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.
Rad51核蛋白丝介导DNA链交换,这是同源重组的关键步骤。复制蛋白A(RPA)可刺激该活性,但前提是在Rad51核蛋白丝形成后再引入RPA。相反,RPA通过预先结合单链DNA(ssDNA)来抑制Rad51核蛋白复合物的形成,但Rad52蛋白可减轻这种抑制作用。在此我们表明,Rad51丝的形成与RPA从ssDNA上的置换是同时发生的。这种置换由Rad51蛋白在ssDNA复合物上的限速成核作用引发,随后丝快速延伸。Rad52蛋白可加速Rad51蛋白对RPA的置换。这种加速可能涉及与Rad51蛋白和RPA的直接相互作用。对Rad52-RPA-ssDNA共复合物的检测表明,该共复合物是置换过程中的一个中间体。