A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis

Masato Kato, Takuhiro Ito, Gerhard Wagner, Tom Ellenberger

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.

Original languageEnglish (US)
Pages (from-to)30554-30562
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number29
DOIs
StatePublished - Jul 16 2004

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DNA Primase
Bacteriophage T7
Bacteriophages
DNA-Directed DNA Polymerase
DNA
Zinc
Aromatic Amino Acids
Mutagenesis
DNA-Binding Proteins
Recycling
Alanine
Catalytic Domain
Nuclear magnetic resonance
Scanning
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis. / Kato, Masato; Ito, Takuhiro; Wagner, Gerhard; Ellenberger, Tom.

In: Journal of Biological Chemistry, Vol. 279, No. 29, 16.07.2004, p. 30554-30562.

Research output: Contribution to journalArticle

Kato, Masato ; Ito, Takuhiro ; Wagner, Gerhard ; Ellenberger, Tom. / A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 29. pp. 30554-30562.
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