Blackwell L J, Borowiec J A
Department of Biochemistry, New York University Medical Center, New York 10016.
Mol Cell Biol. 1994 Jun;14(6):3993-4001. doi: 10.1128/mcb.14.6.3993-4001.1994.
Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.
人复制蛋白A是一种单链DNA（ssDNA）结合蛋白，是真核生物DNA复制和DNA修复系统中的必需因子，并且有人提出它在DNA重组过程中发挥作用。该蛋白也是多种控制复制、转录和细胞生长的蛋白质的相互作用靶点。为了了解hRPA在这些过程中的作用，我们检测了hRPA与特定ssDNA分子的结合情况。采用凝胶迁移实验对ssDNA的长度进行“滴定”，发现hRPA能形成不同的多聚体复合物，这些复合物可通过戊二醛交联检测到。在这些复合物中，hRPA单体在ssDNA上利用的最小结合位点大小为8至10个核苷酸（hRPA8 - 10nt复合物），并且似乎协同结合ssDNA。有趣的是，改变凝胶迁移条件揭示了第二种明显不同的复合物的形成，该复合物以大约30个核苷酸的步长结合ssDNA（hRPA30nt复合物），这种复合物与Kim等人描述的复合物相似（C. Kim、R. O. Snyder和M. S. Wold，《分子细胞生物学》12:3050 - 3059，1992）。hRPA8 - 10nt和hRPA30nt复合物均可在溶液中共存。我们推测，hRPA在DNA代谢中的作用可能通过hRPA以这两种模式结合ssDNA的能力来调节。
