Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.
An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.
D002474:Cell-Free System; D045647:DNA Glycosylases; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism / Q000528:radiation effects; D004926:Escherichia coli / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013604:T-Phages / Q000201:enzymology; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D011740:Pyrimidine Dimers; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C036401:deoxyribopyrimidine endonucleosidase