title: Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.
abstract: Gene 5 protein of bacteriophage T7 is a nonprocessive DNA polymerase. During infection of Escherichia coli, T7 annexes the host protein thioredoxin for use as a processivity factor for T7 DNA polymerase. We describe here a genetic method to investigate the interaction between T7 gene 5 protein and E. coli thioredoxin. The strategy is to use thioredoxin mutants that are unable to support the growth of wild-type T7 phage to select for T7 revertant phage that suppress the defect in thioredoxin. A thioredoxin mutation that replaces glycine at position 74 with aspartic acid fails to support the growth of wild-type T7. This mutation is suppressed by six different mutations within T7 gene 5, each of which results in a single amino acid substitution within gene 5 protein. Three of the suppressor mutations are located within the putative polymerization domain of gene 5 protein, and three are located within the putative 3'-to-5' exonucleolytic domain. Each suppressor mutation alone is necessary and sufficient to confer the revertant phenotype.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
authors.forename: J S
authors.initials: JS
authors.lastname: Himawan
authors.forename: C C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism; D016153:Genes, Suppressor; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000201:enzymology*; D013879:Thioredoxins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D046911:Macromolecular Substances; D014764:Viral Proteins; D013879:Thioredoxins; D004259:DNA-Directed DNA Polymerase