Wyka Iwona M, Dhar Kajari, Binz Sara K, Wold Marc S
Department of Biochemistry, University of Iowa College of Medicine, 51 Newton Road, Iowa City, Iowa 52242-1109, USA.
Biochemistry. 2003 Nov 11;42(44):12909-18. doi: 10.1021/bi034930h.
Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.
人类复制蛋白A（RPA）是一种异源三聚体（70 kDa、32 kDa和14 kDa亚基）的真核单链DNA（ssDNA）结合蛋白，是DNA重组、修复和复制所必需的。人类RPA的三个亚基由六个保守的DNA结合结构域（DBD）组成。缺失和突变研究已确定70 kDa亚基中心区域存在一个高亲和力DNA结合核心，由DBD A和DBD B组成。为了确定每个DBD在DNA结合中的作用，构建并表征了单体和串联DBD A和B结构域嵌合体。单独来看，DBD A和DBD B对ssDNA的内在亲和力非常低。相比之下，串联DBD（AA、AB、BA和BB）以中等至高亲和力结合ssDNA。AA嵌合体对ssDNA的亲和力比其他串联DBD高得多，表明DBD A对ssDNA的内在亲和力高于DBD B。DBD A和DBD B中的RPA-DNA界面相似。进行了突变分析以探究这两个结构域对DNA结合的相对贡献。任一核心DBD中极性残基的突变都会导致RPA复合物对ssDNA的亲和力显著降低。具有成对突变极性残基的RPA复合物的亲和力低于具有单个突变的复合物。当极性突变组合时观察到的亲和力下降表明，多种极性相互作用有助于RPA核心对DNA的亲和力。这些结果表明，RPA-ssDNA相互作用是多个不等价结构域结合的结果。我们的数据与RPA的顺序结合模型一致，其中DBD A负责RPA复合物的定位和初始结合，而DBD A与DBD B一起指导与ssDNA的稳定、高亲和力结合。
