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

Figure 4

Insertion opposite and extension from 3-mA by T7 Sequenase (T7) and Klenow fragment (K) (exo⁻) polymerases. The template strand in the absence (−) or presence (+) of the DFO was treated with 0 or 2.5 mM Me-lex as described in Figure 1. A 5^-labeled primer (Table 1, P25) was used that overlaps with the two 3^-A’s (A-24, A-25) that are not significantly methylated by Me-lex (Figure 1). T7 Sequenase (T7) or Klenow polymerase exo⁻ (K) was added and the reaction initiated by the addition of the four dNTPs (0 or 500 𝜇M) to determine insertion opposite, and extension from, 3-mA at 37°C. The production of extended product is reduced with Me-lex treatment in the presence of the DFO.