title: Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome.
abstract: Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is "unlocked" by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.
authors.affiliation: Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, Pennsylvania 16802, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Xi
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Zhang
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
authors.forename: Jingsong
authors.initials: J
authors.lastname: Yang
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Spiering
authors.forename: Gordon G
authors.initials: GG
authors.lastname: Hammes
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology* / Q000502:physiology; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D009690:Nucleic Acid Conformation; D019384:Nucleic Acid Synthesis Inhibitors; D011485:Protein Binding / Q000502:physiology; D013379:Substrate Specificity; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D019065:Virus Assembly / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D009097:Multienzyme Complexes; D019384:Nucleic Acid Synthesis Inhibitors; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; C069983:gene 43 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases