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PMID: 8394445 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen.

Journal of virology ·Vol. 67 ·No. 9 ·1993-09-00 ·Pages 5206-15

McVey D, Ray S, Gluzman Y, Berger L, Wildeman AG, Marshak DR, Tegtmeyer P

Abstract

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.

MeSH Terms
Amino Acid Sequence Antigens, Polyomavirus Transforming/isolation & purification,metabolism CDC2 Protein Kinase/metabolism DNA Helicases/metabolism DNA Polymerase II/metabolism DNA, Superhelical/isolation & purification,metabolism Deoxyribonuclease I Escherichia coli/metabolism Gene Expression Regulation, Viral HeLa Cells Homeostasis Humans Kinetics Models, Structural Phosphorylation Plasmids Protein Binding Protein Conformation Simian virus 40/metabolism,physiology Threonine Transcription, Genetic Virus Replication
Chemicals
Antigens, Polyomavirus Transforming DNA, Superhelical Threonine CDC2 Protein Kinase DNA Polymerase II Deoxyribonuclease I DNA Helicases
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
McVey D
Department of Microbiology, State University of New York, Stony Brook 11794.
Ray S
Gluzman Y
Berger L
Wildeman A G
Marshak D R
Tegtmeyer P
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.67.9.5206-5215.1993
Published
1993-09-00
Pages
5206-15
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC237918
Subset
IM
Grants
NCI NIH HHS · CA-09176 · United States
NCI NIH HHS · CA-13106 · United States
NCI NIH HHS · CA-18808 · United States
External Links
PubMed source DOI: 10.1128/JVI.67.9.5206-5215.1993 PMC Full Text View journal details
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