title: Assembly and dynamics of Gp59-Gp32-single-stranded DNA (ssDNA), a DNA helicase loading complex required for recombination-dependent replication in bacteriophage T4.
abstract: The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Branagan
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Maher
authors.forename: Scott W
authors.initials: SW
authors.lastname: Morrical
mesh_terms: D017122:Bacteriophage T4 / Q000502:physiology*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D011995:Recombination, Genetic / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C091225:gene 59 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D004265:DNA Helicases