Probing the aminofluorene-induced conformational heterogeneity in a simulated translesion synthesis by 19F NMR and differential scanning calorimetry

TOXI 100

Fengting Liang, liangft@uri.edu and Bongsup Cho, bcho@uri.edu. Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881
We report a NMR and DSC investigation of the aminofluorene (AF)-induced conformational heterogeneity in a simulated translesion synthesis. The DSC results revealed that enthalphic changes are strongly modulated by the nature (i.e., dC or dA) of an incorporated base at the lesion site. Dynamic 19F NMR data showed that conformational clarity is driven by thermal and thermodynamic stabilities, as well as the length of the primer past the lesion site. Similar NMR measurements were made with the E. coli DNA polymerase I Klenow exo- in the presence of a template/primer DNA, a non-cleavable &alpha-methylene dNTP substrate (&alpha-m-CTP or &alpha-m-ATP), and a divalent cation (Mg+2). The results from the thermodynamic/NMR experiments support a model in which the adduct structure at the replication fork exerts a “short-term” memory effect through a long-range DNA-protein interaction. The polymerase NMR data provided insight into the ‘active-site induced' conformational heterogeneity. [NIH CA098296 and P20 RR016457]