title: Simultaneous interactions of bacteriophage T4 DNA replication proteins gp59 and gp32 with single-stranded (ss) DNA. Co-modulation of ssDNA binding activities in a DNA helicase assembly intermediate.
abstract: The T4 gp59 protein is the major accessory protein of the phage's replicative DNA helicase, gp41. gp59 helps load gp41 at DNA replication forks by promoting its assembly onto single-stranded (ss) DNA covered with cooperatively bound molecules of gp32, the T4 single-strand DNA binding protein (ssb). A gp59-gp32-ssDNA ternary complex is an obligatory intermediate in this helicase loading mechanism. Here, we characterize the properties of gp59-gp32-ssDNA complexes and reveal some of the biochemical interactions that occur within them. Our results indicate the following: (i) gp59 is able to co-occupy ssDNA pre-saturated with either gp32 or gp32-A (a truncated gp32 species lacking interactions with gp59); (ii) gp59 destabilizes both gp32-ssDNA and (gp32-A)-ssDNA interactions; (iii) interactions of gp59 with the A-domain of gp32 alter the ssDNA-binding properties of gp59; and (iv) gp59 organizes gp32-ssDNA versus (gp32-A)-ssDNA into morphologically distinct complexes. Our results support a model in which gp59-gp32 interactions are non-essential for the co-occupancy of both proteins on ssDNA but are essential for the formation of structures competent for helicase assembly. The data argue that specific "cross-talk" between gp59 and gp32, involving conformational changes in both, is a key feature of the gp41 helicase assembly pathway.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Lefebvre
authors.forename: M L
authors.initials: ML
authors.lastname: Wong
authors.forename: S W
authors.initials: SW
authors.lastname: Morrical
mesh_terms: D017104:Bacteriophage M13; D017122:Bacteriophage T4; D016172:DNA Fingerprinting; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism* / Q000648:ultrastructure; D004279:DNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D017665:Hydroxyl Radical; D008954:Models, Biological; D008957:Models, Genetic; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D014764:Viral Proteins / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; D017665:Hydroxyl Radical; D004265:DNA Helicases