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PMID: 9199322 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

Molecular and cellular biology ·Vol. 17 ·No. 7 ·1997-07-00 ·Pages 3876-83

Iftode C, Borowiec JA

Abstract

The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.

MeSH Terms
Base Sequence DNA Helicases/metabolism DNA Replication DNA, Single-Stranded/ultrastructure DNA, Viral/ultrastructure DNA-Binding Proteins/metabolism Gene Expression Regulation, Viral Humans Molecular Sequence Data Nucleic Acid Denaturation Proteins/physiology Replication Origin Simian virus 40/genetics Trans-Activators
Chemicals
DNA, Single-Stranded DNA, Viral DNA-Binding Proteins Proteins Trans-Activators replication initiator protein DNA Helicases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Iftode C
Department of Biochemistry and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York 10016, USA.
Borowiec J A
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.17.7.3876
Published
1997-07-00
Pages
3876-83
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC232240
Subset
IM
Grants
NIAID NIH HHS · AI29963 · United States
NCI NIH HHS · P30CA16087 · United States
External Links
PubMed source DOI: 10.1128/MCB.17.7.3876 PMC Full Text View journal details
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