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PMID: 2173787 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome.

Journal of virology ·Vol. 64 ·No. 12 ·1990-12-00 ·Pages 6204-13

Snyder RO, Im DS, Muzyczka N

Abstract

We have demonstrated that when the covalently joined ends of linear adeno-associated virus (AAV) DNA are resolved in vitro, the virus-encoded Rep protein becomes covalently attached to the 5' ends of the DNA. The covalent bond is between a tyrosine residue of the AAV Rep protein and a 5' phosphate of a thymidine residue in the AAV genome. Only the Rep protein encoded by the AAV p5 promoter, Rep68, was capable of becoming covalently attached to the ends of the AAV genome; the Rep proteins encoded by the p19 promoter were not. We also investigated some of the requirements for the complete in vitro resolution reaction. Inhibitor studies suggested that terminal resolution required DNA polymerase delta, ATP, and the deoxyribonucleoside triphosphates but did not require the remaining ribonucleoside triphosphates, DNA polymerase alpha, RNA polymerase II, or topoisomerases I and II. Finally, purified AAV Rep68, when added to the crude cytosol from uninfected HeLa cells, was sufficient for resolution. This suggested that terminal resolution relies on host enzymes and the virus-encoded p5 Rep proteins.

MeSH Terms
Binding Sites DNA Polymerase III DNA Replication DNA, Viral/genetics,metabolism DNA-Directed DNA Polymerase/metabolism Dependovirus/genetics Genes, Viral HeLa Cells/metabolism Humans Models, Genetic Nucleic Acid Hybridization Plasmids Protein Binding Tyrosine Viral Proteins/metabolism
Chemicals
DNA, Viral Viral Proteins Tyrosine DNA Polymerase III DNA-Directed DNA Polymerase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Snyder R O
Department of Microbiology, State University of New York, Stony Brook Medical School 11794-8621.
Im D S
Muzyczka N
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.64.12.6204-6213.1990
Published
1990-12-00
Pages
6204-13
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC248795
Subset
IM
Grants
NCI NIH HHS · 5PO1 CA2814607 · United States
NIAID NIH HHS · AI25530 · United States
NIGMS NIH HHS · R01 GM3572302 · United States
External Links
PubMed source DOI: 10.1128/JVI.64.12.6204-6213.1990 PMC Full Text View journal details
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