TY - JOUR
T1 - 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
AU - Kotapati, Srikanth
AU - Maddukuri, Leena
AU - Wickramaratne, Susith
AU - Seneviratne, Uthpala
AU - Goggin, Melissa
AU - Pence, Matthew G.
AU - Villalta, Peter
AU - Guengerich, F. Peter
AU - Marnett, Lawrence
AU - Tretyakova, Natalia
PY - 2012/11/9
Y1 - 2012/11/9
N2 - The 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′- deoxyadenosine (1,N6-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,N6-propano group on 1,N6-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,N6-γ-HMHP-dA lesions in the presence of humanDNApolymerases (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,N6-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,N6-γ-HMHP- dA and detected large amounts of -1 and 12 deletion products. Taken together, these results indicate that hpol η and κenzymes 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.
AB - The 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′- deoxyadenosine (1,N6-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,N6-propano group on 1,N6-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,N6-γ-HMHP-dA lesions in the presence of humanDNApolymerases (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,N6-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,N6-γ-HMHP- dA and detected large amounts of -1 and 12 deletion products. Taken together, these results indicate that hpol η and κenzymes 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.
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U2 - 10.1074/jbc.M112.396788
DO - 10.1074/jbc.M112.396788
M3 - Article
C2 - 22977231
AN - SCOPUS:84869036510
SN - 0021-9258
VL - 287
SP - 38800
EP - 38811
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -