Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases (original) (raw)

2012, Journal of Biological Chemistry

Background: 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2Ј-deoxyadenosine (1,N 6-␥-HMHP-dA) adducts are formed in DNA by 1,2,3,4-diepoxybutane (metabolite of human carcinogen 1,3-butadiene). Results: hpols and carry out translesion synthesis, incorporating T, G, or A opposite the 1,N 6-␥-HMHP-dA adduct. Conclusion: Translesion bypass of 1,N 6-␥-HMHP-dA adducts by TLS polymerases is error-prone. Significance: This study identifies 1,N 6-␥-HMHP-dA as the DNA adduct potentially responsible for A3 T and A3 C transversions and deletions induced by 1,3-butadiene. The 1,N 6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2deoxyadenosine (1,N 6-␥-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N 6propano group on 1,N 6-␥-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N 6-␥-HMHP-dA lesions in the presence of human DNA polymerases (hpols) ␤, , , and and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol , , and Dpo4 were able to copy the modified template. In contrast, hpol inserted a single base opposite 1,N 6-␥-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol ␤. Single nucleotide incorporation experiments indicated that although hpol , , and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N 6-␥-HMHP-dA and detected large amounts of ؊1 and ؊2 deletion products. Taken together, these results indicate that hpol and enzymes bypass 1,N 6-␥-HMHP-dA lesions in an error-prone fashion, potentially contributing to A3 T and A3 C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.