title: Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA.
abstract: Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Darin
authors.initials: D
authors.lastname: Dolezal
authors.forename: Charles E
authors.initials: CE
authors.lastname: Jones
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Lai
authors.forename: J Rodney
authors.initials: JR
authors.lastname: Brister
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Mueser
authors.forename: Nancy G
authors.initials: NG
authors.lastname: Nossal
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics*; D001665:Binding Sites; D004265:DNA Helicases / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C091225:gene 59 protein, Enterobacteria phage T4; D004265:DNA Helicases