Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine

Abstract

Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.deoxynucleotide kinase ͉ dideoxynucleosides ͉ DNA metabolism ͉ T7 DNA polymerase ͉ thymidine kinase W hen bacteriophage T7 infects Escherichia coli, the host RNA polymerase transcribes T7 early genes, designated class I genes. This group includes the T7 RNA polymerase (gene 1) (1). Approximately 6-15 min after infection, T7 RNA polymerase transcribes a second group of T7 genes, designated class II genes, that encode most of the proteins involved in DNA metabolism. The class II genes include those that encode the helicase/primase (gene 4), the DNA polymerase (gene 5) (1), and a single-stranded DNA-binding protein (gene 2.5) (2). A number of class II genes are not essential under laboratory growth conditions and have not yet been assigned specific functions (1).Little is known about the process of deoxynucleotide metabolism in T7-infected E. coli. T7 derives most of the phosphorus incorporated into its DNA from the breakdown of E. coli DNA (3, 4). The host DNA is degraded to 5Ј-deoxynucleoside monophosphates (dNMPs) by the joint action of the gene 3 endonuclease (5) and gene 6 exonuclease (6). It is not known whether the phage encodes its own enzymes to convert dNMPs to the dNTP precursors for DNA synthesis. E. coli encodes four different dNMP kinases, each specific for one dNMP (7,8). At least one of these kinases, CMP kinase, encoded by cmk, is essential for T7 growth (9). In turn, nucleoside diphosphokinase is the enzyme primarily responsible for conversion of dNDPs to dNTPs (10). Phage T4 encodes its own set of dNMP kinases with specificities that differ from that of the E. coli enzymes, whereas it uses the E. coli nucleoside diphosphokinase to convert the dNDPs to dNTPs (11). Phage T4 also encodes a thymidine kinase (tdk) (12, 13). Neither E. coli nor phage T7 will readily use exogenous thymine for DNA synthesis; in contrast, phage T4 efficiently incorporates exogenous thymine into its DNA (13).One of the interesting properties of T7 DNA polymerase is it...

Characterization of a Nucleotide Kinase Encoded by Bacteriophage T7

“…It is tempting to speculate that the dodecamer plays a functional role in the metal-independent reaction, perhaps forming a protein based compartment (30) in which the critical residues from monomer are either components of the catalytic interfaces or are involved in subunit assembly. We have shown that even a single amino acid alteration in the C-terminal half of gp1.7 renders T7 phage resistant to ddT (1). The crystal structure of this remarkable enzyme should provide insight into the molecular basis of its catalytic activity.…”

“…1B). Furthermore, deletion genetic mapping has shown that the N-terminal half of gp1.7 is not required for conferring sensitivity of T7 phage to dideoxythymidine (1,2). We have also purified gp1.7 under denaturing conditions followed by renaturation to release any tightly bound metal.…”

“…Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction. Gene 1.7 of bacteriophage T7 came to our attention when we found that mutations in gene 1.7 rendered T7 growth on Escherichia coli resistant to exogenous dideoxythymidine (ddT) in the media (1). E. coli can grow in the presence of ddT up to 5 mM, whereas T7 fails to form plaques in the presence of 0.1 mM ddT.…”

“…1 To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA.…”

It Seems Like Only Yesterday

“…The selection was for T7 phage that could grow in media containing dideoxynucleosides on the basis of the predicted phenotype of a T7 DNA polymerase that could not incorporate dideoxythymidine 5 -monophosphate (ddTMP). T7 phage resistant to inhibition by dideoxythymidine were isolated, but all the mutations resided in gene 1.7, a nonessential gene (256). After several years of frustration for others, Ngoc Tran found that the difficulty in purification was the precipitation of gene 1.7 protein at low ionic strength, such as 100 mM NaCl (234).…”