title: 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.
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.
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Srikanth
authors.initials: S
authors.lastname: Kotapati
authors.forename: Leena
authors.initials: L
authors.lastname: Maddukuri
authors.forename: Susith
authors.initials: S
authors.lastname: Wickramaratne
authors.forename: Uthpala
authors.initials: U
authors.lastname: Seneviratne
authors.forename: Melissa
authors.initials: M
authors.lastname: Goggin
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Pence
authors.forename: Peter
authors.initials: P
authors.lastname: Villalta
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
authors.forename: Lawrence
authors.initials: L
authors.lastname: Marnett
authors.forename: Natalia
authors.initials: N
authors.lastname: Tretyakova
mesh_terms: D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D018736:DNA Adducts; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D003839:Deoxyadenosines / Q000494:pharmacology*; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: C550993:1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine; D018736:DNA Adducts; D003839:Deoxyadenosines; D009841:Oligonucleotides; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase