Abstract
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.
Highlights
The economy of proteins involved in the replication of the linear double-stranded DNA chromosome of bacteriophage T7 has made it an attractive model for dissecting the proteinprotein interactions that are essential for coordination of the multiple reactions that occur at a replication fork [1]
The four proteins that account for the basic reactions at the T7 replication fork are T7 gene 5 DNA polymerase, the host Escherichia coli thioredoxin, T7 gene 4 helicase/primase, and the T7 gene 2.5 single-stranded DNA1 binding protein
The 63-kDa gene 4 protein catalyzes the template-directed synthesis of oligoribonucleotides at specific recognition sites on single-stranded DNA (ssDNA), a reaction that is dependent on the presence of the zinc binding motif [18, 22,23,24,25,26]
Summary
The economy of proteins involved in the replication of the linear double-stranded DNA chromosome of bacteriophage T7 has made it an attractive model for dissecting the proteinprotein interactions that are essential for coordination of the multiple reactions that occur at a replication fork [1]. The 56kDa form is translated from an internal initiation codon that is in-frame with the coding sequence for the 63-kDa protein [15] Both forms of the protein have helicase activity, bind ssDNA in the presence of a nucleoside triphosphate, and translocate 5Ј to 3Ј along the DNA strand using the energy of nucleoside 5Јtriphosphate hydrolysis [15,16,17,18]. The 63-kDa gene 4 protein catalyzes the template-directed synthesis of oligoribonucleotides at specific recognition sites on ssDNA, a reaction that is dependent on the presence of the zinc binding motif [18, 22,23,24,25,26]. Inasmuch as the 63-kDa gene 4 protein has both helicase and primase activities, it alone is sufficient to support T7 DNA replication and phage growth [27, 28]
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