Functional Domains of the 70-Kilodalton Subunit of Human Replication Protein A

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• 作者：Xavier V. Gomes and Marc S. Wold
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：August 13, 1996
• 年：1996
• 卷：35
• 期：32
• 页码：10558 - 10568
• 全文大小：705K
• 年卷期：v.35,no.32(August 13, 1996)
• ISSN：1520-4995

文摘

Human replication protein A (RPA) is a single-strandedDNA-binding protein that is composedof subunits of 70, 32, and 14 kDa. This heterotrimeric complex isrequired for multiple processes inDNA metabolism including DNA replication, DNA repair, andrecombination. Previous studies havesuggested that the 616 amino acid, 70-kDa subunit of RPA (RPA70) iscomposed of multiple structural/functional domains. We used a series of N-terminal deletions ofRPA70 to define the boundaries ofthese domains and elucidate their functions. Mutant RPA complexesmissing residues 1-168 of RPA70bound ssDNA with high affinity and supported SV40 replication invitro. In contrast, deletions extendingbeyond residue 168 showed a decreased affinity for ssDNA and wereinactive in SV40 DNA replication.When residues 1-381 were deleted, the resulting truncated RPA70was unable to bind ssDNA but stillformed a stable complex with the 32- and 14-kDa subunits of RPA.Thus, the C-terminal domain ofRPA70 is both necessary and sufficient for RPA complex formation.These data indicate that RPA70 iscomposed of three functional domains: an N-terminal domain that isnot required for ssDNA binding orSV40 replication, a central DNA-binding domain, and a C-terminal domainthat is essential for subunitinteractions. For all mutant complexes examined, bothphosphorylation of the 32-kDa subunit of RPAand the ability to support T antigen-dependent, origin-dependent DNAunwinding correlated with ssDNAbinding activity.