Effect of N3-Methyladenine and an Isosteric Stable Analogue on DNA Polymerization
Figure 2
The 3-mA lesion blocks DNA polymerization by T7 Sequenase (T7) and Klenow fragment exo− (K) polymerase. (a) DNA template (100 nM) was treated with 0 or 2.50 mM Me-lex for 12 hours at 4°C in the presence or absence of the DFO: 3-mA forms at A-26 and A-27 only when the DFO is present (Figure 1). The runup polymerization assay was initiated by raising incubation temperature to 24 or 37°C and the addition of -Cy5-primer (200 nM) and dNTPs (0 or 100 M). Polymerization experiments with T7 Sequenase and and Klenow fragment (exo−) were run for 5 or 10 minutes. (b) The Me-lex (0, 0.6, 1.25 or 2.50 mM) treated DNA was used to extend -Cy5-primer in the absence (−) or presence of 100 M dNTPs for 5 or 10 minutes at 37°C using T7 Sequenase. The polymerase is blocked prior to insertion opposite positions-26 or -27, which are heavily methylated by Me-lex (Figure 1).
