DNA substrates and 5'-end labeling

Oligodeoxyribonucleotides were 5'-³²P-phosphorylated with T4 polynucleotide kinase and purified by 20% polyacrylamide/7.0 M urea gel electrophoresis as described [42], followed by electro-elution and precipitation with 2% solution of LiClO₄ in acetone. The precipitated oligodeoxyribonucleotides were dissolved in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (TE buffer). Complementary oligonucleotides were annealed by heating (90°C, 5 min) a solution of equimolar amounts, followed by slow cooling-down to room temperature, to form the double-stranded DNA substrates containing nick (DNApF and DNAp), uracil (DNA-U) or 3-hydroxy-2-hydroxymethyltetrahydrofuran (DNA-F). The structures of DNA duplexes used are summarized in Table 1.

Abbreviations

U-uracil

F-3-hydroxy-2-hydroxymethyltetrahydrofuran

pF* = 3-hydroxy-2-hydroxymethyltetrahydrofuran with 5'-phosphate

p** = phosphate

^FABGC, exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-monophosphate.