title: Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies.
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) (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.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, NC 27710, USA.
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Zhang
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Spiering
authors.forename: Michael A
authors.initials: MA
authors.lastname: Trakselis
authors.forename: Faoud T
authors.initials: FT
authors.lastname: Ishmael
authors.forename: Jun
authors.initials: J
authors.lastname: Xi
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
authors.forename: Gordon G
authors.initials: GG
authors.lastname: Hammes
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D017122:Bacteriophage T4 / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D000255:Adenosine Triphosphate
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