화학공학소재연구정보센터
Journal of the American Chemical Society, Vol.129, No.40, 12108-12108, 2007
Probing the thermodynamics of aminofluorene-induced translesion DNA synthesis by differential scanning calorimetry
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 (Delta Delta G degrees = 1.7 kcal/mol, Delta Delta H degrees = 9.1 kcal/mol), no such thermodynamic advantage was observed with the FAF lesion at n (Delta Delta G degrees similar to 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.