Figure 2.

Cleavage activity of hNTH1 on duplex oligonucleotides with or without 5-foU. (A) Cleavage of 5-foU-containing oligonucleotides by purified hNTH1. The duplex oligonucleotide, 5-foU/A (lanes 1–4) or T/A (lanes 5–7), was incubated at 37°C for 20 min without protein (lanes 1 and 5) or with 24 ng hNTH1 (lanes 2 and 6), 25 ng E.coli Nth (lanes 4 and 7) or 24 ng heat-inactivated hNTH1 (lane 3). The inactivation of hNTH1 was performed by heating at 100°C for 10 min. After the reaction was terminated, the products were separated by denaturing 20% PAGE on gels containing 7 M urea. (B) Formation of Schiff base intermediate with 5-foU-containing oligonucleotides by hNTH1. The duplex oligonucleotide substrates were incubated with or without enzyme at 37°C for 5 min in the presence of 50 mM NaBH₄. The amounts of hNTH1 and Nth in the reaction mixture were 200 and 100 ng, respectively.