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PMID: 15793585 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Nature structural & molecular biology ·Vol. 12 ·No. 4 ·2005-04-00 ·Pages 332-9

Arunkumar AI, Klimovich V, Jiang X, Ott RD, Mizoue L, Fanning E, Chazin WJ

Abstract

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.

MeSH Terms
Antibodies, Monoclonal/immunology,pharmacology Binding Sites DNA/genetics,metabolism DNA Primers/biosynthesis,genetics DNA Repair DNA Replication/physiology DNA-Binding Proteins/antagonists & inhibitors,chemistry,immunology,metabolism Humans Models, Molecular Mutation/genetics Nuclear Magnetic Resonance, Biomolecular Protein Binding Protein Structure, Tertiary Replication Protein A Simian virus 40/genetics,growth & development Virus Replication/drug effects,physiology
Chemicals
Antibodies, Monoclonal DNA Primers DNA-Binding Proteins RPA1 protein, human Replication Protein A DNA
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Arunkumar Alphonse I
Department of Biochemistry, Vanderbilt University, Nashville Tennessee 37232-8725 USA.
Klimovich Vitaly
Jiang Xiaohua
Ott Robert D
Mizoue L
Fanning Ellen
Chazin Walter J
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Article Info
Journal
Nature structural & molecular biology
Abbr.
Nat Struct Mol Biol
ISSN
1545-9993
DOI
10.1038/nsmb916
Published
2005-04-00
Epub
2005-00-27
Pages
332-9
Language
English
Region
United States
NLM ID
101186374
PMCID
PMC2600586
Subset
IM
Grants
NIGMS NIH HHS · R01 GM065484 · United States
NIGMS NIH HHS · R01 GM065484-01A1 · United States
NIGMS NIH HHS · R01 GM065484-02 · United States
Databases
PDB
1Z1D
External Links
PubMed source DOI: 10.1038/nsmb916 PMC Full Text View journal details
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