Role of Protein-Protein Interactions in the Function of Replication Protein A (RPA): RPA Modulates the Activity of DNA Polymerase b

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• 作者：Katherine A. Braun ; Ye Lao ; Zhigang He ; C. James Ingles ; and Marc S. Wold
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：July 15, 1997
• 年：1997
• 卷：36
• 期：28
• 页码：8443 - 8454
• 全文大小：399K
• 年卷期：v.36,no.28(July 15, 1997)
• ISSN：1520-4995

文摘

Replication Protein A (RPA) from human cells is a stable complexof 70-, 32-, and 14-kDasubunits that is required for multiple processes in DNA metabolism.RPA binds with high affinity tosingle-stranded DNA and interacts with multiple proteins, includingproteins required for the initiation ofSV40 DNA replication, DNA polymerase s/gifchars/alpha.gif" BORDER=0> and SV40 large T antigen.We have used a series of mutantderivatives of RPA to map the regions of RPA required for specificprotein-protein interactions andhave examined the roles of these interactions in DNA replication.T antigen, DNA polymerase s/gifchars/alpha.gif" BORDER=0> and theactivation domain of VP16 all have overlapping sites of interaction inthe N-terminal half (residues 1-327)of the 70-kDa subunit of RPA. In addition, the interaction sitefor DNA polymerase s/gifchars/alpha.gif" BORDER=0> is composed oftwo functionally distinct regions, one (residues 1-~170) whichstimulates polymerase activity and asecond (residues ~170-327) which increases polymerase processivity.In the latter, both the directprotein-protein interaction and ssDNA-binding activities of RPA wereneeded for RPA to modulatepolymerase processivity. We also found that SV40 T antigeninhibited the ability of RPA to increaseprocessivity of DNA polymerase s/gifchars/alpha.gif" BORDER=0>, suggesting that this activity ofRPA may be important for elongationbut not during the initiation of DNA replication. DNA polymerases/gifchars/alpha.gif" BORDER=0>, but not T antigen also interactedwith the 32- and/or 14-kDa subunits of RPA, but these interactions didnot seem to effect polymeraseactivity.