The oligomeric T4 primase is the functional form during replication

Jingsong Yang, Jun Xi, Zhihao Zhuang, Stephen Benkovic

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Replisome DNA primases are responsible for the synthesis of short RNA primers required for the initiation of repetitive Okazaki fragment synthesis on the lagging strand during DNA replication. In bacteriophage T4, the primase (gp61) interacts with the helicase (gp41) to form the primosome complex, an interaction that greatly stimulates the priming activity of gp61. Because gp41 is hexameric, a question arises as to whether gp61 also forms a hexameric structure during replication. Several results from this study support such a structure. Titration of the primase/single-stranded DNA binding followed by fluorescence anisotropy implicated a 6:1 stoichiometry. The observed rate constant, kcat, for priming was found to increase with the primase concentration, implicating an oligomeric form of the primase as the major functional species. The generation of hetero-oligomeric populations of the hexameric primase by controlled mixing of wild type and an inactive mutant primase confirmed the oligomeric nature of the most active primase form. Mutant primases defective in either the N- or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits. Collectively, these results provide strong evidence for the functional oligomerization of gp61. The potential roles of gp61 oligomerization during lagging strand synthesis are discussed.

Original languageEnglish (US)
Pages (from-to)25416-25423
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number27
DOIs
StatePublished - Jul 8 2005

Fingerprint

DNA Primase
Oligomerization
Bacteriophage T4
Bacteriophages
Fluorescence Polarization
Single-Stranded DNA
DNA Replication
Titration
Stoichiometry
Rate constants
Catalyst activity
Catalytic Domain
Anisotropy
Fluorescence

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Yang, Jingsong ; Xi, Jun ; Zhuang, Zhihao ; Benkovic, Stephen. / The oligomeric T4 primase is the functional form during replication. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 27. pp. 25416-25423.
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The oligomeric T4 primase is the functional form during replication. / Yang, Jingsong; Xi, Jun; Zhuang, Zhihao; Benkovic, Stephen.

In: Journal of Biological Chemistry, Vol. 280, No. 27, 08.07.2005, p. 25416-25423.

Research output: Contribution to journalArticle

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