title: Control of helicase loading in the coupled DNA replication and recombination systems of bacteriophage T4.
abstract: The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.
authors: From the Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors: Amy M
authors: AM
authors: Branagan
authors: Jenny A
authors: JA
authors: Klein
authors: Christian S
authors: CS
authors: Jordan
authors: Scott W
authors: SW
authors: Morrical
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases
keywords: Bacteriophage; D-loop; DNA Helicase; DNA Repair; DNA Replication; Homologous Recombination; Mediator; Origin; R-loop; ssDNA-binding Protein