Open AccessAssembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies
Author(s) -
Zhiquan Zhang,
Michelle M. Spiering,
Michael A. Trakselis,
Faoud T. Ishmael,
Jun Xi,
Stephen J. Benkovic,
Gordon G. Hammes
Publication year - 2005
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0500327102
Subject(s) - replisome , dna , primase , helicase , dnab helicase , dna replication , dna clamp , biophysics , biology , prokaryotic dna replication , dnag , bacteriophage , single stranded binding protein , biochemistry , microbiology and biotechnology , chemistry , dna binding protein , rna , control of chromosome duplication , circular bacterial chromosome , gene , reverse transcriptase , escherichia coli , transcription factor
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) (MgATPgammaS) 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.