Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4.
The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.
D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects*; D005796:Genes; D005814:Genes, Viral; D005816:Genetic Complementation Test; D005838:Genotype; D007700:Kinetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000528:radiation effects*; D014466:Ultraviolet Rays
D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases