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PMID: 8392624 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

Journal of virology ·Vol. 67 ·No. 8 ·1993-08-00 ·Pages 4992-5002

Moarefi IF, Small D, Gilbert I, Höpfner M, Randall SK, Schneider C, Russo AA, Ramsperger U, Arthur AK, Stahl H

Abstract

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.

MeSH Terms
Alanine Amino Acid Sequence Animals Antigens, Polyomavirus Transforming/genetics,metabolism,ultrastructure Baculoviridae Binding Sites Cyclins/metabolism DNA Helicases/genetics,metabolism,ultrastructure DNA Replication DNA, Viral/metabolism,ultrastructure Humans Kinetics Microscopy, Electron Moths Mutagenesis, Site-Directed Phosphorylation Protein Kinases/metabolism Simian virus 40/genetics,metabolism Threonine Transfection
Chemicals
Antigens, Polyomavirus Transforming Cyclins DNA, Viral Threonine Protein Kinases DNA Helicases Alanine
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Moarefi I F
Institute for Biochemistry, Munich, Germany.
Small D
Gilbert I
Höpfner M
Randall S K
Schneider C
Russo A A
Ramsperger U
Arthur A K
Stahl H
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.67.8.4992-5002.1993
Published
1993-08-00
Pages
4992-5002
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC237887
Subset
IM
Grants
NCI NIH HHS · CA-16519 · United States
NCI NIH HHS · CA-40414 · United States
External Links
PubMed source DOI: 10.1128/JVI.67.8.4992-5002.1993 PMC Full Text View journal details
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