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'-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.
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States.
V G
VG
Vaidyanathan
Bongsup P
BP
Cho
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D005449:Fluorenes / Q000737:chemistry*; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013237:Stereoisomerism; D013816:Thermodynamics
D018736:DNA Adducts; D005449:Fluorenes; C031838:N-(deoxyguanosin-8-yl)-2-aminofluorene; D004247:DNA; D004256:DNA Polymerase I; D003849:Deoxyguanosine