Replication Protein A Interactions with DNA. 1. Functions of the DNA-Binding and Zinc-Finger Domains of the 70-kDa Subunit

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• 作者：André ; P. Walther ; Xavier V. Gomes ; Ye Lao ; Chang Geun Lee ; and Marc S. Wold
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：March 30, 1999
• 年：1999
• 卷：38
• 期：13
• 页码：3963 - 3973
• 全文大小：156K
• 年卷期：v.38,no.13(March 30, 1999)
• ISSN：1520-4995

文摘

Human replication protein A (RPA) is a multiple subunit single-stranded DNA-binding proteinthat is required for multiple processes in cellular DNA metabolism. This complex, composed of subunitsof 70, 32, and 14 kDa, binds to single-stranded DNA (ssDNA) with high affinity and participates inmultiple protein-protein interactions. The 70-kDa subunit of RPA is known to be composed of multipledomains: an N-terminal domain that participates in protein interactions, a central DNA-binding domain(composed of two copies of a ssDNA-binding motif), a putative (C-X₂-C-X₁₃-C-X₂-C) zinc finger, and aC-terminal intersubunit interaction domain. A series of mutant forms of RPA were used to elucidate theroles of these domains in RPA function. The central DNA-binding domain was necessary and sufficientfor interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and part ofthe N-terminal domain, were needed for optimal ssDNA-binding activity. The role of aromatic residuesin RPA-DNA interactions was examined. Mutation of any one of the four aromatic residues shown tointeract with ssDNA had minimal effects on RPA activity, indicating that individually these residues arenot critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex,reduced ssDNA-binding activity, and eliminated activity in DNA replication.