The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase

J Biol Chem. 1997 Jul 18;272(29):18425-33. doi: 10.1074/jbc.272.29.18425.


The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wild-type protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage T7 / enzymology*
  • Bacteriophage T7 / genetics
  • Base Sequence
  • Binding Sites
  • DNA Primase
  • DNA Replication
  • DNA-Directed DNA Polymerase / metabolism*
  • Genetic Complementation Test
  • Kinetics
  • Macromolecular Substances
  • Models, Structural
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • RNA Nucleotidyltransferases / chemistry
  • RNA Nucleotidyltransferases / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Templates, Genetic


  • Macromolecular Substances
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • DNA Primase
  • RNA Nucleotidyltransferases
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase