FIG. 1.

Incorporation of oxidized nucleotides by DNA polymerases. The incorporation of 2-OH-dATP (A) and 8-OH-dGTP (B) into DNA by DNA Pol IV and KF exo⁻ of E. coli was assayed as described previously (28). Cy3-conjugated primer, annealed to the template at a 1:1 ratio (0.1 μM), was incubated with DNA Pol IV (0.1 μM) or KF exo⁻ (0.02 U), and then 50 μM 2-OH-dATP (A) or 50 μM 8-OH-dGTP (B) was added. No other dNTPs were added to the reaction mixtures. All the reactions were carried out at room temperature for 30 min. The reaction products were analyzed on 15% denatured polyacrylamide gels, and the bands were visualized using a Molecular Imager FX Pro system (Bio-Rad, Richmond, CA). The oligonucleotide sequences of the primer and template were 5′-Cy3-CGCGCGAAGACCGGTTAC-3′ and 5′-GAAGGGATCCTTAAGACNGTAACCGGTCTTCGCGCG-3′, respectively, for 2-OH-dATP and 5′-Cy3-CGGAGCTCGGTCGGCGTCTGCGTC and 5′-AGCCGCAGGAGNGACGCAGACGCCGACCGAGCTCCG-3′, respectively, for 8-OH-dGTP (N = A, C, G, or T). Parts of the sequences of the primer and template are shown. The unlabeled lanes on the left indicate the positions of Cy3-labeled primers without extension.