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The oligomeric T4 primase is the functional form during replication
Journal article   Open access   Peer reviewed

The oligomeric T4 primase is the functional form during replication

Jingsong Yang, Jun Xi, Zhihao Zhuang and Stephen J Benkovic
The Journal of biological chemistry, v 280(27), pp 25416-25423
08 Jul 2005
PMID: 15897200
url
https://doi.org/10.1074/jbc.M501847200View
Published, Version of Record (VoR) Open

Abstract

Protein Structure, Tertiary DNA Helicases - chemistry Viral Proteins - chemistry DNA Primase - metabolism Viral Proteins - genetics Bacteriophage T4 - genetics Viral Proteins - metabolism DNA Helicases - metabolism DNA Replication - physiology Mutagenesis Bacteriophage T4 - enzymology DNA Primase - genetics DNA - biosynthesis Fluorescence Polarization DNA Helicases - genetics DNA Primase - chemistry
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, k(cat), 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.

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Biochemistry & Molecular Biology
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