(I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [ γ - 32 P] 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 <t>DNA</t> 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 <t>SacI</t> 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. ∗ U: uracil; ∗∗ AP: apurinic/apyrimidinic.
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