Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

Abstract

N2,3-Ethenoguanine (N2,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N2,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N2,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N2,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N2,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N2,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N2,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N2,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N2,3-ϵG.

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

“…Taken together, these data suggest that hpol is more efficient than hpol in replication bypass in the context of the sequence and adduct studied in this work. The order of bypass efficiency, hpol Ͼ hpol , is consistent with previously reported values for 1, (67), and N 6 -dA-(OH) 2 butyl-GSH (29). Additional information was obtained from capillary HPLC-ESI Ϫ -MS/MS sequencing of extension products (Table 2).…”

“…These enzymes catalyze error-prone bypass of 8-oxo-dG (72,73), abasic sites (72-74), 1,N 6 -(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2Ј-deoxyadenosine (66), 1,N 6 -etheno-dA (49), and N 2 ,3-etheno-dG (67). hpol also reads past bulky benzo[a]pyrene-N 2 -dG (72) in an errorprone manner, while hpol erroneously bypasses 2-acetylaminofluorene-dG (73,74).…”

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

“…As observed previously for C5-thymine lesions (36), hPol was more efficient than hPol . Higher catalytic efficiency of hPol than hPol is consistent with previous reports for other bulky nucleobase lesions such as exocyclic adducts (35,(71)(72)(73) and DNA-glutathione conjugates (74). Unlike C5-thymine-peptide lesions, which induce high numbers of frameshift and base substitution mutations upon primer extension in the presence of hPol and hPol (36), C7-G conjugated DPCs were not miscoding.…”

Replication past the Butadiene Diepoxide-Derived DNA Adduct S-[4-(N6-Deoxyadenosinyl)-2,3-dihydroxybutyl]glutathione by DNA Polymerases

“…The order of bypass efficiency, Pol T7 ~ Pol η > Dpo4 > Pol κ > Pol ι (Figure 1), is similar to the reported bypass efficiency past N 2 ,3-ethenoguanine by these polymerases. 32,42 Compared with Pol κ and Pol ι, Pol η exhibited higher bypass efficiency past the adducts 1, N 6 -ethenoadenine 43,44 and 1, N 6 -(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2´-deoxyadenosine (1, N 6 -γ-HMHP-dA) adduct (formed from DEB), 45 which has been proposed to contribute to mutagenicity of BD. 16 However, Pol ι showed some single base incorporation opposite 1, N 6 -γ-HMHP-dA, 45 and did not extend primer well opposite N 2 ,3-ethenoguanine.…”

“…16 However, Pol ι showed some single base incorporation opposite 1, N 6 -γ-HMHP-dA, 45 and did not extend primer well opposite N 2 ,3-ethenoguanine. 42 The ability of Pol T7, a high-fidelity processive polymerase, to efficiently bypass these large adducts positioned at a Watson-Crick pairing site, is surprising, in light of previous work with N 2 dG adducts. 33,46 …”