Figure 7.

Reaction of PFBHA and abasic DNA. A. The oxyamine adduct PFBHA-DNA is the product of the reaction between PFBHA and abasic DNA. This reaction is analogous to the well-characterized reaction of methoxyamine with abasic DNA^48–50 but has the advantage that the larger PFBHA adduct is more easily separated from the abasic DNA substrate on a gel. B. PFBHA reacts with 2 or 2/3 to form PFBHA-DNA. No adduct forms in the absence of PFBHA. Reactions contained 0.25 µM DNA and were carried out at 37 °C and pH 6.5 with or without 1.5 mM PFBHA. Samples were reduced with NaBH₄ before separation on a 20% denaturing polyacrylamide gel. This image displays only the region of the gel where the 35mer and the PFBHA-35mer appear; A full image of the same gel (Figure S3) shows that ICL formation is minimal in reactions of duplex 2/3 when PFBHA is present, because PFBHA outcompetes the exocyclic amine for reaction with the abasic site at the concentrations of PFBHA used. C. The k_obs for formation of PFBHA-DNA increases with PFBHA concentration in duplex 2/3 reactions. Values of k_obs at each PFBHA concentration were determined from exponential fits to the data and are plotted in D. D. Linear fits for the dependence of k_obs on PFBHA concentration give second order rate constants for the reaction of PFBHA with 2 of 0.06 mM⁻¹min⁻¹ and with 2/3 of 0.14 mM⁻¹min⁻¹.