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PMID: 1328866 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.

Molecular and cellular biology ·Vol. 12 ·No. 11 ·1992-11-00 ·Pages 4883-95

Virshup DM, Russo AA, Kelly TJ

Abstract

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.

MeSH Terms
Antigens, Polyomavirus Transforming/metabolism Base Sequence DNA Helicases/metabolism DNA Replication DNA, Viral/biosynthesis,genetics Molecular Sequence Data Mutation Phosphoprotein Phosphatases/metabolism Phosphorylation Protein Phosphatase 2 Simian virus 40/genetics
Chemicals
Antigens, Polyomavirus Transforming DNA, Viral Phosphoprotein Phosphatases Protein Phosphatase 2 DNA Helicases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Virshup D M
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Russo A A
Kelly T J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.12.11.4883-4895.1992
Published
1992-11-00
Pages
4883-95
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC360421
Subset
IM
Grants
NIAID NIH HHS · AI-31657 · United States
NCI NIH HHS · CA-40414 · United States
NIDDK NIH HHS · DK-01528 · United States
External Links
PubMed source DOI: 10.1128/mcb.12.11.4883-4895.1992 PMC Full Text View journal details
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