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Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies
Journal article   Open access

Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies

Zhiquan Zhang, Michelle M Spiering, Michael A Trakselis, Faoud T Ishmael, Jun Xi, Stephen J Benkovic and Gordon G Hammes
Proceedings of the National Academy of Sciences - PNAS, v 102(9), pp 3254-3259
01 Mar 2005
PMID: 15728347
url
https://doi.org/10.1073/pnas.0500327102View
Published, Version of Record (VoR) Open

Abstract

Biological Sciences DNA replication fluorescence-resonance energy transfer fluorescence microscopy
Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5′- O -(3-thiotriphosphate) (MgATPγS) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41·DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the primosome on the lagging strand of DNA at a replication fork.

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Collaboration types
Domestic collaboration
Web of Science research areas
Biochemistry & Molecular Biology
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