Uracil-DNA glycosylase inhibitor of bacteriophage PBS2: cloning and effects of expression of the inhibitor gene in Escherichia coli.
The uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 was cloned, and the effects of this inhibitor on Escherichia coli cells that contain uracil-DNA glycosylase activity were determined. A PBS2 genomic library was constructed by inserting EcoRI restriction fragments of PBS2 DNA into a plasmid pUC19 vector. The library was used to transform wild-type (ung+) E. coli, and the presence of the functional inhibitor gene was determined by screening for colonies that supported growth of M13mp19 phage containing uracil-DNA. A clone was identified that carried a 4.1-kilobase EcoRI DNA insert in the vector plasmid. Extracts of cells transformed with this recombinant plasmid lacked detectable uracil-DNA glycosylase activity and contained a protein that inhibited the activity of purified E. coli uracil-DNA glycosylase in vitro. The uracil-DNA glycosylase inhibitor expressed in these E. coli was partially purified and characterized as a heat-stable protein with a native molecular weight of about 18,000. Hence, we conclude that the PBS2 uracil-DNA glycosylase inhibitor gene was cloned and that the gene product has properties similar to those from PBS2-infected Bacillus subtilis cells. Inhibitor gene expression in E. coli resulted in (i) a weak mutator phenotype, (ii) a growth rate similar to that of E. coli containing pUC19 alone, (iii) a sensitivity to the antifolate drug aminopterin similar to that of cells lacking the inhibitor gene, and (iv) an increased resistance to the lethal effects of 5-fluoro-2'-deoxyuridine. These physiological properties are consistent with the phenotypes of other ung mutants.
D000630:Aminopterin / Q000494:pharmacology; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004926:Escherichia coli / Q000235:genetics; D005467:Floxuridine / Q000494:pharmacology; D005786:Gene Expression Regulation; D005814:Genes, Viral; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors*; D010641:Phenotype; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D005467:Floxuridine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D000630:Aminopterin