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PMID: 6813861 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 79 ·No. 18 ·1982-09-00 ·Pages 5522-6

Peñalva MA, Salas M

Abstract

Incubation of extracts of phi 29-infected Bacillus subtilis with [alpha-32P]dATP produced a labeled protein having the electrophoretic mobility of p3, the 5'-terminal protein of phi 29 DNA. The reaction product was resistant to treatment with micrococcal nuclease, phosphatase, and RNases A and T1 and sensitive to proteinase K. Incubation of the 32P-labeled protein with piperidine under conditions in which the phi 29 DNA-protein p3 linkage is hydrolyzed released 5'-dAMP. The reaction with [alpha-32P]dATP was strongly inhibited by anti-p3 serum and required the preence of phi 29 DNA-protein p3 complex; no reaction took place with proteinase K-treated phi29 DNA. These results, together with those of acid hydrolysis and partial proteolysis, indicated that a covalent complex between protein p3 and 5'-dAMP is formed in vitro. The initiation complex (protein p3-dAMP) formed in the presence of 0.5 microM [alpha-32P]dATP can be elongated by addition of 40 microM dNTPs. Treatment with piperidine of the product elongated in the presence of 2',3'-dideoxycytidine 5'-triphosphate released the expected oligonucleotides, 9 and 12 bases long, taking into account the sequence at the left and right DNA ends, respectively.

MeSH Terms
Bacillus subtilis/genetics Bacteriophages/genetics DNA Replication Deoxyadenine Nucleotides/metabolism Protein Binding Templates, Genetic Viral Proteins/genetics,metabolism Virus Replication
Chemicals
Deoxyadenine Nucleotides Viral Proteins 2'-deoxy-5'-adenosine monophosphate
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Peñalva M A
Salas M
References (33)
33 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.79.18.5522
Published
1982-09-00
Pages
5522-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC346936
Subset
IM
Grants
NIGMS NIH HHS · 1 R01 GM27242-02 · United States
External Links
PubMed source DOI: 10.1073/pnas.79.18.5522 PMC Full Text View journal details
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