Sibenaller Z A, Sorensen B R, Wold M S
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242, USA.
Biochemistry. 1998 Sep 8;37(36):12496-506. doi: 10.1021/bi981110+.
Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homologues are heterotrimeric complexes with subunits of approximately 70, approximately 32, and approximately 14 kDa. While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low cooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides) and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing the high-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties. In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weak ssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variations in the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two proteins.
复制蛋白A（RPA）是一种多亚基单链DNA结合（ssDNA）蛋白，是细胞DNA代谢所必需的。在所有已检测的真核生物中都鉴定出了RPA同源物。所有同源物都是异源三聚体复合物，其亚基大小约为70 kDa、约32 kDa和约14 kDa。虽然RPA同源物在进化上是保守的，但它们在功能上并不等同。为了更好地理解RPA同源物之间的功能差异，我们分析了来自人类细胞和芽殖酵母酿酒酵母（分别为hRPA和scRPA）的RPA的DNA结合参数。酵母和人类的RPA都以高亲和力和低协同性结合ssDNA。然而，scRPA具有更大的封闭结合位点（45个核苷酸对34个核苷酸），并且对寡聚胸腺嘧啶的亲和力高于hRPA。含有来自70 kDa亚基的高亲和力DNA结合结构域的hRPA和scRPA突变形式具有几乎相同的DNA结合特性。相比之下，来自酵母和人类RPA的32 kDa和14 kDa亚基的亚复合物具有较弱的ssDNA结合活性。然而，酵母和人类亚复合物的结合常数分别比RPA异源三聚体低3个和大于6个数量级。我们得出结论，RPA的32 kDa和14 kDa亚基活性的差异是scRPA和hRPA的ssDNA结合特性变化的原因。这些数据还表明，hRPA和scRPA与ssDNA的结合模式不同，这可能导致这两种蛋白质之间的功能差异。
