Kim C, Snyder R O, Wold M S
Department of Biochemistry, University of Iowa, Iowa City 52242.
Mol Cell Biol. 1992 Jul;12(7):3050-9. doi: 10.1128/mcb.12.7.3050-3059.1992.
Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.
复制蛋白A（RP-A；也称为复制因子A和人单链DNA结合蛋白）是一种单链DNA结合蛋白，在体外猿猴病毒40 DNA复制中是必需的。从人和酵母细胞中分离出的RP-A是一种非常稳定的复合物，由3个亚基（70、32和14 kDa）组成。我们分析了人和酵母RP-A的DNA结合特性，以便更好地了解它们在DNA复制中的作用。人RP-A对单链DNA具有高亲和力，对RNA和双链DNA具有低亲和力。RP-A对单链DNA的表观亲和常数在10(9) M-1范围内。RP-A的结合位点大小约为30个核苷酸，且不协同结合。RP-A与单链DNA的结合部分依赖于序列。人RP-A对嘧啶的亲和力比对嘌呤的亲和力高约50倍。酵母RP-A的结合特性与人类蛋白相似。酵母和人RP-A都分别优先结合到同源复制起点（ARS307或猿猴病毒40复制起点）富含嘧啶的链上。这种不对称结合表明RP-A可能在DNA复制起始过程中起直接作用。
