Insights into hRPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome

• Published in: Nature structural & molecular biology Vol. 12; no. 4; pp. 332 - 339
• Main Authors: Fanning, Ellen, Chazin, Walter J, Arunkumar, Alphonse I, Klimovich, Vitaly, Jiang, Xiaohua, Ott, Robert D, Mizoue, L
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.04.2005
• Subjects: Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - pharmacology; Binding Sites; Cellular proteins; Chromatography; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - metabolism; DNA polymerase; DNA Primers - biosynthesis; DNA Primers - genetics; DNA Repair; DNA replication; DNA Replication - physiology; DNA-Binding Proteins - antagonists & inhibitors; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - immunology; DNA-Binding Proteins - metabolism; Genetic aspects; Humans; Models, Molecular; Monoclonal antibodies; Mutation; Mutation - genetics; Nuclear Magnetic Resonance, Biomolecular; Physiological aspects; Protein Binding; Protein Structure, Tertiary; Proteins; Replication Protein A; Simian virus 40; Simian virus 40 - genetics; Simian virus 40 - growth & development; Structure; SV40 (Virus); Virus Replication - drug effects; Virus Replication - physiology; Yeast; United States
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb916

Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen (Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA-binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.