Repair of benzo[a]pyrene diol epoxide damaged bacteriophage T7 DNA determined by survival of phage made by in vitro packaging.
DNA from bacteriophage T7 was treated with benzo[a]pyrene diol epoxide (BPDE) and the number of covalently bound adducts per T7 genome was determined. BPDE treated T7 DNA was then incubated in an in vitro DNA packaging system so as to form infective T7 phage. The observed reduced survival of these phage measured with Escherichia coli uvrA- indicator bacteria showed that the BPDE treated DNA was in fact utilized by the in vitro packaging system and that the resulting phage contained DNA damage caused by in vitro exposure to BPDE. T7 DNA damage by BPDE was also incubated in an in vitro DNA repair system that used partially purified uvrABC proteins from E. coli. Alkaline sucrose gradient analysis demonstrated that nicks were introduced into the damaged DNA and that these incisions were repaired to yield nearly intact DNA molecules of about the size of a T7 genome. Encapsulation of the repaired DNA with the packaging system yielded phage that showed higher survival than the unrepaired control when plated on uvrA- indicator bacteria.
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D001580:Benzopyrenes / Q000494:pharmacology*; D002499:Centrifugation, Density Gradient; D004260:DNA Repair; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D013604:T-Phages / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D001580:Benzopyrenes; D004279:DNA, Viral; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide