Sequence Effects on Translesion Synthesis of an Aminofluorene–DNA Adduct: Conformational, Thermodynamic, and Primer Extension Kinetic Studies

The DNA sequence effect is an important structural factor for determining the extent and nature of carcinogen-induced mutational and repair outcomes. In this study, we used two 16-mer template sequences, TG*A [d­(5′-CTTCT<u>TG*A</u>CCTCATTC-3′)] and CG*A [d­(5′-CTTCT<u>CG*A</u>CCTCATTC-3′)], to study the impact of the 5′-flanking nucleotide (T vs C) on aminofluorene (AF)-induced stacked (S)/major groove (B)/wedge (W) conformational heterogeneity during a simulated translesion synthesis. In addition, we probed the sequence effect on nucleotide insertion efficiencies catalyzed by the Klenow fragment (exonuclease-deficient) of DNA polymerase I. Our ¹⁹F NMR/ICD/DSC results showed that AF in the CG*A duplex sequence adopts a greater population of S-conformer than the TG*A sequence. We found that the S conformer of CG*A thermodynamically favors insertion of A over C at the lesion site (<i>n</i>). Significant stalling occurred at both the prelesion (<i>n</i> – 1) and lesion (<i>n</i>) sites; however, the effect was more persistent for the S conformer of CG*A than TG*A at the lesion site (<i>n</i>). Kinetics show that relative nucleotide insertion frequencies (<i>f</i>_ins) were greater for TG*A than the S conformer of CG*A for either dCTP or dATP at the lesion site (<i>n</i>), and the insertion rate was significantly reduced at immediate upstream base pairs (<i>n</i>, <i>n</i> + 1). Taken together, the results provide insight into how the mutagenic AF could exhibit an S/B/W equilibrium in the active site of a polymerase, causing different mutations. This work represents a novel structure–function relationship in which adduct structure is directly linked to nucleotide insertion efficiency in a conformation-specific manner during translesion DNA synthesis.