title: Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.
abstract: We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Ngoc Q
authors.initials: NQ
authors.lastname: Tran
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Tabor
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA ccr@hms.harvard.edu.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D017123:Bacteriophage T7 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004279:DNA, Viral / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D054306:Dideoxynucleotides / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D057845:Pyrimidine Phosphorylases / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D054306:Dideoxynucleotides; D029968:Escherichia coli Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D013942:Thymine Nucleotides; D014764:Viral Proteins; D057845:Pyrimidine Phosphorylases; D013937:Thymidine Kinase; D004259:DNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate; D013936:Thymidine