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PMID: 3863101 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 82 ·No. 19 ·1985-10-00 ·Pages 6404-8

Blanco L, Salas M

Abstract

A system that replicates bacteriophage phi 29 DNA with protein p3 covalently attached to the two 5' ends, using as the only proteins the phi 29 DNA polymerase and the terminal protein, is described. Restriction analysis of the 32P-labeled DNA synthesized in vitro showed that all phi 29 DNA fragments were labeled. Analysis by alkaline sucrose gradient centrifugation of the DNA labeled during a 10-min pulse showed that, after a 20-min chase, about half of the DNA molecules had reached apparently full-length phi 29 DNA (approximately equal to 18,000 nucleotides). Ammonium ions strongly stimulated phi 29 DNA-protein p3 replication, the effect being due to stimulation of the initiation reaction. ATP was not required for phi 29 DNA-protein p3 replication, either in the initiation or elongation steps. The results show that the phi 29 DNA polymerase functions, not only in the formation of the p3-dAMP covalent initiation complex but also in the elongation of the latter, as the only DNA polymerase to produce full-length phi 29 DNA.

MeSH Terms
Adenosine Monophosphate/metabolism Adenosine Triphosphate/metabolism Bacteriophages/enzymology,genetics DNA Replication DNA, Viral/biosynthesis DNA-Directed DNA Polymerase/metabolism Viral Proteins/metabolism,physiology Virus Replication
Chemicals
DNA, Viral Viral Proteins terminal protein, Bacillus phage phi29 Adenosine Monophosphate Adenosine Triphosphate DNA-Directed DNA Polymerase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Blanco L
Salas M
References (30)
30 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.82.19.6404
Published
1985-10-00
Pages
6404-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC390724
Subset
IM
Grants
NIGMS NIH HHS · 5 R01 GM27242-05 · United States
External Links
PubMed source DOI: 10.1073/pnas.82.19.6404 PMC Full Text View journal details
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