Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population

Figure 1

(I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [γ-³²P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. : uracil; : apurinic/apyrimidinic.