HUMAN REPLICATION PROTEIN-A BINDS SINGLE-STRANDED-DNA IN 2 DISTINCT COMPLEXES
HUMAN REPLICATION PROTEIN-A BINDS SINGLE-STRANDED-DNA IN 2 DISTINCT COMPLEXES
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DOI：
10.1128/mcb.14.6.3993
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发表时间：
1994-06-01
影响因子：
5.3
通讯作者：
BOROWIEC, JA
中科院分区：
文献类型：
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作者：
BLACKWELL, LJ;BOROWIEC, JA
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，并被认为在DNA重组过程中起作用。该蛋白质也是控制复制、转录和细胞生长的多种蛋白质相互作用的靶标。为了理解hRPA在这些过程中的作用，我们检查了hRPA与定义的ssDNA分子的结合。采用“滴定”ssDNA长度的凝胶位移测定，发现hRPA形成可通过戊二醛交联检测到的独特的多聚体复合物。在这些复合物中，hRPA的单体利用ssDNA上8至10个核苷酸的最小结合位点大小（hRPA（8- 10 nt）复合物），并且似乎协同结合ssDNA。有趣的是，凝胶位移条件的改变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个核苷酸的步骤结合ssDNA（hRPA（30 nt）复合物），该复合物类似于Kim等人（C.金河，巴西-地O. Snyder和M. S. Wold，Mol. Cell. 12：3050-3059，1992）。hRPA（8- 10 nt）和hRPA（30 nt）复合物均可在溶液中共存。我们推测hRPA在DNA代谢中的作用可能是通过hRPA在这两种模式下结合ssDNA的能力来调节的。
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 hRPA(8-10nt) 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 hRPA(30nt) 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 hRPA(8-10nt) and hRPA(30nt) 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.