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

Abstract

1,2,3,4-Diepoxybutane (DEB), a metabolite of the carcinogen butadiene, has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially A:T to G:C in Salmonella typhimurium TA1535 (Chem. Res. Toxicol. 23, 1544 (2010)) and Escherichia coli TRG8 cells (Chem. Res. Toxicol. 25, 1522 (2012)). We previously identified S-[4-(N6-deoxyadenosinyl)-2,3-dihydroxybutyl]GSH (N6dA-(OH)2butyl-GSH) as a major adduct in the reaction of S-(2-hydroxy-3,4-epoxybutyl)glutathione (DEB-GSH conjugate) with nucleosides and calf thymus DNA and in vivo in livers of mice and rats treated with DEB (Chem. Res. Toxicol. 25, 706 (2012)). For investigation of the miscoding potential of the major DEB-GSH conjugate-derived DNA adduct (N6dA-(OH)2butyl-GSH) and the effect of GSH conjugation on replication of DEB, extension studies were performed in duplex DNA substrates containing the site specifically-incorporated N6dA-(OH)2butyl-GSH adduct, N6-(2,3,4-trihydroxybutyl)deoxyadenosine adduct (N6dA-butanetriol), or unmodified deoxyadenosine (dA) by human DNA polymerases (Pol) η, ι, and κ, bacteriophage polymerase T7, and Sulfolobus solfataricus polymerase Dpo4. Although dTTP incorporation was the most preferred addition opposite the N6dA-(OH)2butyl-GSH adduct, N6dA-butanetriol adduct, or unmodified dA by all polymerases, dCTP misincorporation frequency opposite N6dA-(OH)2butyl-GSH was significantly higher than that opposite the N6dA-butanetriol adduct or unmodified dA by Pol κ or Pol T7. LC-MS/MS analysis of full-length primer extension products confirmed that Pol κ or Pol T7 incorporated the incorrect base C opposite N6dA-(OH)2butyl-GSH lesion. These results indicate the relevance of GSH-containing adducts for the A:T to G:C mutations produced by DEB.

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.…”

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

“…Previous investigations have revealed that the ability of DNA polymerases to bypass DNA-peptide conjugates is dependent on the lesion size, the attachment site within the DNA, and polymerase identity (10,(21)(22)(23)(24)(25)(26)(27)(28)(29). Lloyd and co-workers (26) have reported that human polymerase (hpol) and its Escherichia coli orthologue pol IV were able to catalyze error-free primer extension past DNA templates containing tetra-and dodecapeptides conjugated to the N 2 position of guanine via a trimethylene linker.…”

“…Yamanaka et al (27) proposed that small major groove DPC adducts have sufficient conformational flexibility to be accommodated within the active site of TLS polymerases without disturbing primer-template-enzyme interactions, although the corresponding minor groove adducts block replication. More recently, Guengerich and co-workers (29) reported that human polymerases and , as well as bacterial polymerases pol T7 and DPO4, were capable of replicating DNA containing S- [4-(N 6 -deoxyadenosinyl)-2,3-dihydroxybutyl] glutathione adducts (N 6 -dA-(OH) 2 butyl-GlyCys-␥Glu). However, to our knowledge, no systematic studies have been performed to examine replication bypass of DPCs conjugated to pyrimidine bases on DNA, despite their potential significance in vivo.…”

“…In the resulting primer-template complexes, the 3Ј-primer terminus is positioned immediately prior to the lesion site (Ϫ1 primer, Scheme 2A). Initial single nucleotide incorporation assays were conducted to determine what nucleotides can be inserted opposite the adducted site by human translesion synthesis polymerases, hpol and , which were selected due to their documented ability to catalyze replication past other bulky DNA lesions (26,29). The resulting primer-template duplexes were incubated with recombinant polymerases in the presence of individual dNTPs for 0 -90 min.…”

“…Multiple nucleotide insertions observed in the single nucleotide incorporation experiments (Fig. 1) may be due to the low fidelity of these two TLS polymerases (29,41,42).…”