Gene 4 of bacteriophage T7 encodes two proteins, a 63 kDa and a colinear 56 kDa protein. The coding sequence of the 56 kDa protein begins at the residues encoding an internal methionine located 64 amino acids from the N‐terminus of the 63 kDa protein. The 56 kDa gene 4 protein is a helicase and the 63 kDa gene 4 protein is a helicase and a primase. The unique 7 kDa N‐terminus of the 63 kDa gene 4 protein is essential for primer synthesis and contains sequences with homology to a Cys4 metal binding motif, Cys‐X2‐Cys‐X17‐Cys‐X2‐Cys. The zinc content of the 63 kDa gene 4 protein is 1.1 g‐atom/mol protein, while the zinc content of the 56 kDa gene 4 protein is < 0.01, as determined by atomic absorption spectrometry. A bacteriophage deleted for gene 4, T7 delta 4‐1, is incapable of growing on Escherichia coli strains that contain plasmids expressing gene 4 proteins with single amino acid substitutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(‐7)). Primase containing a substitution of the third Cys for Ser has been overexpressed in E. coli and purified to homogeneity. This mutant primase cannot catalyze template‐directed synthesis of oligoribonucleotides although it is able to catalyze the synthesis of random diribonucleotides in a template‐independent fashion. The mutant primase has reduced helicase activity although it catalyzes single‐stranded DNA‐dependent hydrolysis of dTTP at rates comparable with wild type primase. The zinc content of the mutant primase is 0.5 g‐atom/mol protein.

Escherichia coli optAI, a mutant unable to support the growth of T7 phage containing mutations in gene 1.2, contains reduced amounts of dGTP. Extracts of E. coli optAl catalyze the hydrolysis of dGTP at a rate 50-fold greater than do extracts of E. coli optA+ . The dGTPase responsible for the increased hydrolysis has been purified to apparent homogeneity. Purification of the protein is facilitated by its high affinity for single-stranded DNA. By using this purification scheme an identical dGTPase has been purified from E. coli optA+. The purified proteins catalyze the hydrolysis of dGTP to yield deoxyguanosine and tripolyphosphate. The products of hydrolysis, chromatographic properties, denatured molecular mass of 56 kDa, N-terminal amino acid sequence, substrate specificity, and heat inactivation indicate that the proteins purified from optAl and from optA + cells are identical and identify the enzyme as the deoxyguanosine 5'-triphosphate triphosphohydrolase purified to homogeneity from wild-type E. coli Isolation of the optAl mutant of Escherichia coli was based on the assumption that the products of some genes of phage 17 were not essential for growth because one or more host proteins could substitute for them (1). E. coli optAl demonstrated no obvious phenotype other than its inability to support the growth of 17 gene 1.2 mutants; the optAl mutation is located at 3.6 min on the E. coli linkage map (1). Gene 1.2 of phage T7 is located at position 15.37 on the 17 chromosome (2, 3) and encodes a 10-kDa protein. Expression of gene 1.2 protein is subject to a form of posttranscriptional regulation (4); expression is dependent on the pattern of cleavage of mRNA at the RNase III recognition site immediately following gene 1.2.Characterization of E. coli optAl infected with T7 1.2 mutant phage showed that 17 DNA synthesis terminated prematurely and the DNA was degraded (1). Additional insight came from studies with T4 phage mutants (5, 6). Certain mutations (antimutator phenotype) in T4 gene 43 (DNA polymerase) as well as mutations in the dexA gene (exonuclease) render it unable to grow on E. coli optAl.What is the biochemical basis for these diverse effects of the optAl mutation on phage growth? We have shown that E. coli optAl cells have lower levels of dGTP (by a factor of 5) than do optA + cells (7) Can a deficiency in dGTP levels also explain the inability of T4 dexA and T4 CB120 mutants to grow on E. coli optAl (5, 6)? The dexA gene encodes an oligonucleotidase that, though not essential for growth, does participate in the degradation of host DNA (9). We have proposed that when the level of dGTP is low, as in an optAl host, degradation of the host DNA by the dexA nuclease is required to yield dGMP (7). T4 CB120 carries a mutation in gene 43, the gene for T4 DNA polymerase, that gives rise to an antimutator phenotype (10). The T4 CB120 polymerase has a 10-to 100-fold increased rate of nucleotide turnover (11), a parameter that reflects the hydrolysis of incorporated nucleotides during DNA synthesis. Suc...

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis (1-3). The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain and a primase domain, located in the C-terminal and N-terminal halves of the protein, respectively (4). The T7 helicase functions as a hexamer and has been used as a model to study ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds to single-stranded DNA (ssDNA) 3 as a hexamer and translocates 5Ј to 3Ј along the DNA strand using the energy of hydrolysis of dTTP (5-7). T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however, dTTP hydrolysis is optimally coupled to DNA unwinding (5).Most hexameric helicases use rATP to fuel translocation and unwind DNA (3). T7 helicase does hydrolyze rATP but with a 20-fold higher K m as compared with dTTP (5, 8). It has been suggested that T7 helicase actually uses rATP in vivo where the concentration of rATP is 20-fold that of dTTP in the Escherichia coli cell (8). However, hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA (9). Other ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor hydrolysis observed is not coupled to DNA unwinding (8). Furthermore, Patel et al. (10) found that the form of T7 helicase found in v...

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