Probing the thermodynamics of aminofluorene-induced translesion DNA synthesis by differential scanning calorimetry
Date of Original Version
Differential scanning calorimetry (DSC) was used to investigate the thermodynamic contribution to replication fidelity and specificity associated with translesion synthesis across FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene, a fluorine-tagged DNA adduct derived from the carcinogen 2-aminofluorene. As a control, insertion of matched nucleoside dC at the primer terminus (n) and subsequent extensions (n + 1 to n + 6) resulted in an incremental increase in thermodynamic parameters. In contrast, incorporation of dC opposite FAF-dG and subsequent extensions up to n + 2 showed little change in thermodynamics. A similar thermodynamic stalling was observed for a control template primer containing a G:A mismatch at n and subsequent Watson-Crick primer extensions. The thermodynamic paucity generated by either a lesion or a mismatch was not localized at the replication fork but extended to 5′-downstream n + 2 sites, thus providing an explanation for the short-term memory effects observed with replicative polymerases. Interestingly, FAF modification did not alter the overall DSC profiles of the G:A mismatch template primer and, in fact, resulted in thermal stabilization. Entropy around the lesion site appears to play a critical role in the adduct-induced increase in thermodynamic stability. While addition of matched nucleoside dC at n was thermodynamically favored over the presence of a mismatched dA (ΔΔG° = 1.7 kcal/mol, ΔΔH° = 9.1 kcal/mol), no such thermodynamic advantage was observed with the FAF lesion at n (ΔΔG° ∼0 kcal/mol). These equilibrium thermodynamic results provide insight into the most prevalent mismatch (i.e., G → T transversion mutations) induced by this lesion. However, kinetic effects undoubtedly play a key role in the processing of this bulky lesion, and the nature of the polymerase is likely to govern and to determine the balance between kinetic and thermodynamic effects. Copyright © 2007 American Chemical Society.
Journal of the American Chemical Society
Liang, Fengting, and Bongsup P. Cho. "Probing the thermodynamics of aminofluorene-induced translesion DNA synthesis by differential scanning calorimetry." Journal of the American Chemical Society 129, 40 (2007): 12108-12109. doi:10.1021/ja075271p.