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

Abstract

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'-CTTCTTG*ACCTCATTC-3')] and CG*A [d(5'-CTTCTCG*ACCTCATTC-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 (19)F 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 (n). Significant stalling occurred at both the prelesion (n - 1) and lesion (n) sites; however, the effect was more persistent for the S conformer of CG*A than TG*A at the lesion site (n). Kinetics show that relative nucleotide insertion frequencies (f(ins)) were greater for TG*A than the S conformer of CG*A for either dCTP or dATP at the lesion site (n), and the insertion rate was significantly reduced at immediate upstream base pairs (n, n + 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.

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

“…According to the structural characterization of Bf (40), FAF at the templating position of the binary complex is sterically constrained, deterring rotation about the glycosidic bond to an anti-B-type conformer to form either a Watson-Crick base pair with an incoming dCMPcPP or possibly a Hoogsteen pair with dAMPcPP. The primer extension data obtained by using a similar template-primer exhibited a preferential incorporation of correct dCTP opposite the FAF lesion followed by a small percentage of dATP insertion (33). Consequently, the major (green) and the upfield minor (red) signals in Fig.…”

“…have shown previously (12,32,33,35,39) that purine bases 3Ј to the FAF-adducted site favors the S-conformation, whereas the presence of pyrimidine bases 5Ј to the lesion site favored the major groove B-conformer (36). In full duplexes, a 5Ј-T favors a higher percentage of the major groove B-type conformer, whereas a 5Ј-C favors the stacked conformation.…”

“…Quenched samples were heated to 95°C for 5 min and immediately cooled on ice. The products were resolved on a denaturing polyacrylamide gel and quantified as described previously (33).…”

Conformational Insights into the Lesion and Sequence Effects for Arylamine-Induced Translesion DNA Synthesis: 19F NMR, Surface Plasmon Resonance, and Primer Kinetic Studies

“…Signals were assigned according to published procedures based on relative chemical shifts, dynamic NMR signal patterns, and H/D isotope effects. 33,35 19 F shielding is a hallmark of the van der Waals interactions and the ring current effects caused by the carcinogen moiety within the stacked and bulge duplexes (S-type conformation). We have studied a number of 19 F NMR spectra of arylamine-modified duplexes (including FABP and FAF) in various sequence settings and in all cases found that the fluorine of the S-type conformer resonates upfield relative to that of the external binding B-type conformer.…”

“…34,35 The reactions were performed at 20 °C. For the unmodified sequence, reactions were performed in a shorter time period of 1 min for correct nucleotide incorporation and for up to 45 min in the case of incorrect nucleotide incorporation.…”