THE ACIDIC CARBOXYL-TERMINUS OF THE BACTERIOPHAGE-T7 GENE-4 HELICASE PRIMASE INTERACTS WITH T7 DNA-POLYMERASE/

The gene 4 proteins of bacteriophage T7 provide both primase and helic ase activities at the replication fork. Efficient DNA replication requ ires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal do main of the gene 4 protein is required for interaction with T7 DNA pol ymerase 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 the se 17 residues results in a gene 4 protein that cannot support the gro wth of T7 phage. The purified mutant gene 4 protein has wild-type leve ls of both helicase and primase activities; however, DNA synthesis cat alyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtain ed with wildtype protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysi s, the protein cannot form a stable complex with the DNA polymerase. T he 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 implicat ions for the mechanisms coupling leading and lagging strand DNA synthe sis at the T7 replication fork.