The 32- and 14-Kilodalton Subunits of Replication Protein A Are Responsible for Species-Specific Interactions with Single-Stranded DNA

详细信息    查看全文

• 作者：Zita A. Sibenaller ; Brenda R. Sorensen ; and Marc S. Wold
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：September 8, 1998
• 年：1998
• 卷：37
• 期：36
• 页码：12496 - 12506
• 全文大小：142K
• 年卷期：v.37,no.36(September 8, 1998)
• ISSN：1520-4995

文摘

Replication protein A (RPA) is a multisubunit single-stranded DNA-binding (ssDNA) proteinthat is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotesexamined. All homologues are heterotrimeric complexes with subunits of ~70, ~32, and ~14 kDa.While RPA homologues are evolutionarily conserved, they are not functionally equivalent. To gain abetter understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the budding yeast Saccharomyces cerevisiae (hRPAand scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and lowcooperativity. However, scRPA has a larger occluded binding site (45 nucleotides versus 34 nucleotides)and a higher affinity for oligothymidine than hRPA. Mutant forms of hRPA and scRPA containing thehigh-affinity DNA-binding domain from the 70-kDa subunit had nearly identical DNA binding properties.In contrast, subcomplexes of the 32- and 14-kDa subunits from both yeast and human RPA had weakssDNA binding activity. However, the binding constants for the yeast and human subcomplexes were 3and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. Weconclude that differences in the activity of the 32- and 14-kDa subunits of RPA are responsible for variationsin the ssDNA-binding properties of scRPA and hRPA. These data also indicate that hRPA and scRPAhave different modes of binding to ssDNA, which may contribute to the functional disparities betweenthe two proteins.