Effect of N3-Methyladenine and an Isosteric Stable Analogue on DNA Polymerization

Figure 3

The effect of 3-mA on DNA polymerization by Polη. The DNA template (100 nM) was treated with 0, 1.25 or 2.5 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 DFO is present (Figure 1). The runup polymerization was initiated by raising incubation temperature to 37°C and the addition of 5^-Cy5-primer (200 nM) and dNTPs (0 or 100 𝜇M). Polymerization experiments were run for 10 or 20 minutes. The polymerase is blocked at the methylated adenines, which is also reflected in the level of extended product. Extension is efficient if the DFO is not present during the methylation step (Scheme 1).