Enzymatic analysis of oligonucleotides containing cyclobutane pyrimidine photodimers with a cleaved intradimer phosphodiester linkage.
Our recent studies indicate that enzymatic hydrolysis of the intradimer phosphodiester linkage constitutes an early reaction in processing UV light-induced cis-syn-cyclobutane pyrimidine dimers in cultured human fibroblasts. Before characterizing the resultant modified dimer sites in cellular DNA, it is necessary to establish experimental conditions that can distinguish backbone-nicked from intact dimers. We thus constructed a model substrate, i.e. p(dT) 10 <> p(dT)10 containing a dimer with a ruptured sugar-phosphate bond, and determined the products of its reaction with snake venom phosphodiesterase and alkaline phosphatase, an enzymatic digestion mixture known to release dimers from UV-treated poly(dA).poly(dT) within trinucleotides with the photoproduct intact at the 3'-end (d-TpT<p>T). The model substrate was prepared by (i) end labeling p(dT)9 using terminal deoxynucleotidyltransferase and [3H]thymine-labeled TTP; and (ii) annealing the chromatographically purified p(dT)10 oligomers to poly(dA) followed by UV (290 nm)-induced ligation. Photoligated 20-mers with one radioactive and modified internal dimer were isolated and enzymatically digested. High performance liquid chromatographic analysis of the reaction products revealed a novel trithymidylate with its backbone severed at the 3'-terminus (d-TpT<>dT), demonstrating that this procedure could discriminate between intact and modified dimers. The procedure was then exploited to show that (i) Escherichia coli DNA photolyase can monomerize, albeit inefficiently, backbone-ruptured dimers; and (ii) phage T4 polynucleotide kinase can catalyze the phosphorylation of d-TpT<>dT, thus facilitating the development of a sensitive postlabeling assay suitable for modified dimer detection under biologically relevant conditions.
Liuzzi
M
M
Department of Medicine, Cross Cancer Institute, Edmonton, Alberta, Canada.
Paterson
M C
MC
D004249:DNA Damage; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D066298:In Vitro Techniques; D010710:Phosphates; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000032:analysis*
D010710:Phosphates; D011740:Pyrimidine Dimers; D004253:DNA Nucleotidylexotransferase; D010727:Phosphoric Diester Hydrolases; D004255:Deoxyribodipyrimidine Photo-Lyase