Brill S J, Bastin-Shanower S
Department of Molecular Biology, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855, USA.
Mol Cell Biol. 1998 Dec;18(12):7225-34. doi: 10.1128/MCB.18.12.7225.
Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2. Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B. The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D). The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast. Interestingly, the amino-terminal portion of domain C contains a putative Cys4-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32. We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc. The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.
复制蛋白A（RPA）是真核生物的异源三聚体单链DNA（ssDNA）结合蛋白（SSB），在其最大亚基RPA1中包含两个同源的ssDNA结合结构域（A和B），在其第二大亚基RPA2中包含第三个结构域。在此我们报告，酿酒酵母RPA1含有一个先前未检测到的ssDNA结合结构域（结构域C），它与结构域A和B串联排列。结构域C的羧基末端部分与结构域A和B以及RPA2中结合ssDNA的区域（结构域D）具有序列相似性。已知与ssDNA碱基堆积的结构域A和B中的芳香族残基在结构域C中保守，并且与结构域A一样，其中一个对于酵母的生存力是必需的。有趣的是，结构域C的氨基末端部分包含一个类似于另一种原核SSB即T4 gp32的推定的Cys4型锌结合基序。我们证明结构域C的ssDNA结合活性对半胱氨酸修饰具有独特的敏感性，但与gp32一样，ssDNA结合并不严格依赖于锌。因此，RPA异源三聚体由至少四个ssDNA结合结构域组成，并展现出细菌和噬菌体SSB的特征。
