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

Zhiquan Zhang, Michelle M. Spiering, Michael A. Trakselis, Faoud T. Ishmael, Jun Xi, Stephen J. Benkovic, Gordon G. Hammes

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3254-3259
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number9
DOIs
StatePublished - Mar 1 2005

All Science Journal Classification (ASJC) codes

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