Abstract
Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to single-stranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Delta26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations.
Highlights
The single-stranded DNA1-binding protein of bacteriophage T7, encoded by gene 2.5 of the phage, is essential for T7 DNA replication and for growth of the phage (1)
E. coli single-stranded DNA-binding (SSB) protein stimulated oligoribonucleotide synthesis ϳ2-fold, whereas T4 gp32 protein inhibited synthesis by Ͼ10fold (13). To further characterize these interactions, we examined the ability of WT gp2.5 and E. coli SSB protein to affect gp4-catalyzed primer synthesis at various concentrations
Effect of Variants of Gp2.5 on Primer Synthesis Catalyzed by Gp4 —As in the earlier experiments with gp2.5 and E. coli SSB protein, we examined the effect of gp2.5-⌬26C and gp2.5-F232L on oligoribonucleotide synthesis catalyzed by gp4 on a 70-mer oligonucleotide containing a primase recognition site (Fig. 7)
Summary
The single-stranded DNA (ssDNA)1-binding protein of bacteriophage T7, encoded by gene 2.5 of the phage, is essential for T7 DNA replication and for growth of the phage (1). Chimeric proteins in which the acidic C termini of either E. coli SSB protein or T4 gene 32 protein were substituted for the C terminus of gp2.5 support the growth of T7 phage lacking gene 2.5. They dimerized and physically interacted with gp and T7 DNA polymerase. Using a random mutagenesis procedure, we recently isolated a number of gp2.5s having single amino acid substitutions that could not support T7 growth (20) Whereas studies with these altered proteins led to the identification of sites that affected DNA binding (7, 21) and homologous base pairing (11), none of the altered proteins appeared to be defective in interactions
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