Sugiyama T, New J H, Kowalczykowski S C
Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665, USA.
Proc Natl Acad Sci U S A. 1998 May 26;95(11):6049-54. doi: 10.1073/pnas.95.11.6049.
Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.
酿酒酵母中的同源重组严重依赖于RAD52的功能。在体外，Rad52蛋白优先结合单链DNA（ssDNA），介导互补ssDNA的退火，并刺激Rad51蛋白介导的DNA链交换。复制蛋白A（RPA）是一种ssDNA结合蛋白，对重组过程也至关重要。在此我们报告，Rad52蛋白可促使RPA-ssDNA复合物退火，而该复合物在其他情况下无法退火。Rad52蛋白促进退火的能力取决于ssDNA底物和ssDNA结合蛋白的类型。RPA可促进但会减缓Rad52蛋白介导的寡核苷酸退火。相比之下，RPA对于更长的质粒大小的DNA退火几乎是必不可少的，但对聚（dT）和聚（dA）的退火影响很小，聚（dT）和聚（dA）是没有二级结构的相对较长的DNA分子。这些结果表明，RPA在Rad52蛋白介导的退火中的一个作用是消除DNA二级结构。然而，大肠杆菌ssDNA结合蛋白和人RPA都不能替代此反应，这表明RPA在此过程中还有第二个作用，这个作用需要特定的RPA-Rad52蛋白相互作用。与非同源ssDNA复合的RPA会抑制退火，但人RPA-ssDNA复合物则不会，这一发现证实了这一观点。最后，我们提出了一个酵母双链DNA断裂修复早期步骤的模型。
