title: Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4.
abstract: Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTPγS analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 61. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well.
authors.affiliation: Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.
authors.forename: Davis
authors.initials: D
authors.lastname: Jose
authors.forename: Steven E
authors.initials: SE
authors.lastname: Weitzel
authors.forename: Debra
authors.initials: D
authors.lastname: Jing
authors.forename: Peter H
authors.initials: PH
authors.lastname: von Hippel
mesh_terms: D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry; D017122:Bacteriophage T4 / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry*; D019915:DNA Primase / Q000737:chemistry*; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D046911:Macromolecular Substances / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D008956:Models, Chemical; D011489:Protein Denaturation; D013816:Thermodynamics; D014461:Ultracentrifugation / Q000379:methods; D014759:Viral Envelope Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D046911:Macromolecular Substances; D014759:Viral Envelope Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D004247:DNA; D019915:DNA Primase; D004265:DNA Helicases