Abstract
The 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-γ-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(6)-propano group on 1,N(6)-γ-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)-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) β, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N(6)-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol β. Single nucleotide incorporation experiments indicated that although hpol η, κ, 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)-γ-HMHP-dA and detected large amounts of -1 and -2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N(6)-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.
Highlights
1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene
Five translesion synthesis polymerases (TLS) DNA polymerases are primarily involved in translesion synthesis in humans as follows: hpol , hpol , hpol , and Rev1 from the Y family and hpol from the B family of polymerases (6 – 8)
Primer Extension Studies in the Presence of All Four dNTPs— Our initial studies investigated the ability of DNA polymerases to bypass 1,N6-␥-HMHP-dA lesions in the presence of all four dNTPs. 1,N6-␥-HMHP-dA was site- incorporated into an 18-mer template (5Ј-TCATXGAATCCTTCCCCC-3Ј, where X ϭ 1,N6-␥-HMHP-dA), which was annealed to a 13-mer primer (5Ј-GGGGGAAGGATTC-3Ј)
Summary
Materials—Full-length recombinant hpol , hpol , and hpol enzymes used for steady-state kinetics experiments were obtained from Enzymax (Lexington, KY). The assays were conducted at 37 °C in buffered solutions containing 50 mM Na-MOPS (pH 7.5), 50 mM NaCl, 5 mM DTT, 100 g/ml BSA, 10% glycerol (v/v) with 50 nM radiolabeled primer-template complexes, and 5 nM polymerase enzymes (except for Dpo, which was used at 25 nM and hpol , 2 units). Primer Extension Assays—32P-End-labeled primer-template complexes containing either unmodified dA or 1,N6-␥HMHP-dA at position X of the template strand were incubated with DNA polymerases as described above in the presence of all four dNTPs (500 M) for 0 – 60 min. HPLC-ESI-MS/MS Analysis of Primer Extension Products from DNA Polymerase Reactions—Oligodeoxynucleotide 18-mers (5Ј-TCATXGAATCCTTCCCCC-3Ј, where X ϭ dA or (R,S)-1,N6-␥-HMHP-dA) were annealed to the 13-mer primer (5Ј-GGGGGAAGGAUTC-3Ј) (100 pmol each) in 20 l of 50 mM NaCl to form primer-template complexes. Expected CID fragmentation patterns of oligonucleotides were obtained using the Mongo Oligo mass calculator version 2.06 available from the Mass Spectrometry Group of Medicinal Chemistry, University of Utah
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