title: The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation.
abstract: The gene 4 protein of bacteriophage T7, a functional hexamer, comprises DNA helicase and primase activities. Both activities depend on the unidirectional movement of the protein along single-stranded DNA in a reaction coupled to the hydrolysis of dTTP. We have characterized dTTPase activity and hexamer formation for the full-length gene 4 protein (gp4) as well as for three carboxyl-terminal fragments starting at residues 219 (gp4-C219), 241 (gp4-C241), and 272 (gp4-C272). The region between residues 242 and 271, residing between the primase and helicase domains, is critical for oligomerization of the gene 4 protein. A functional TPase active site is dependent on oligomerization. During native gel electrophoresis, gp4, gp4-C219, and gp4-C241 migrate as oligomers, whereas gp4-C272 is monomeric. The steady-state k(cat) for dTTPase activity of gp4-C272 increases sharply with protein concentration, indicating that it forms oligomers only at high concentrations. gp4-C219 and gp4-C241 both form a stable complex with gp4, whereas gp4-C272 interacts only weakly with gp4. Measurements of surface plasmon resonance indicate that a monomer of T7 DNA polymerase binds to a dimer of gp4, gp4-C219, or gp4-C241 but to a monomer of gp4-C272. Like the homologous RecA and F(1)-ATPase proteins, the oligomerization domain of the gene 4 protein is adjacent to the amino terminus of the NTP-binding domain.
authors.affiliation: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: S
authors.initials: S
authors.lastname: Guo
authors.forename: S
authors.initials: S
authors.lastname: Tabor
authors.forename: C C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001704:Biopolymers; D002384:Catalysis; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006868:Hydrolysis; D010446:Peptide Fragments / Q000302:isolation & purification; D011755:Pyrophosphatases / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D001704:Biopolymers; D017931:DNA Primers; D010446:Peptide Fragments; D013942:Thymine Nucleotides; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D011755:Pyrophosphatases; C023117:thymidine-triphosphatase; D004265:DNA Helicases; C024157:thymidine 5'-triphosphate