Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome.
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.
Arunkumar
Alphonse I
AI
Department of Biochemistry, Vanderbilt University, Nashville Tennessee 37232-8725 USA.
Klimovich
Vitaly
V
Jiang
Xiaohua
X
Ott
Robert D
RD
Mizoue
L
L
Fanning
Ellen
E
Chazin
Walter J
WJ
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000096:biosynthesis / Q000235:genetics; D004260:DNA Repair; D004261:DNA Replication / Q000502:physiology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051716:Replication Protein A; D013539:Simian virus 40 / Q000235:genetics / Q000254:growth & development*; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA