Human carcinogen 1,3-butadiene (BD) induces large numbers of mutations at A:T base pairs. However, the structural identities of nucleobase adducts responsible for these genetic changes remain to be established. BD is metabolically activated to mutagenic epoxides 3,4-epoxy-1-butene (EB) and 1, 2, 3, 4-diepoxybutane (DEB). EB and DEB alkylate adenine residues of DNA to give N6-(2-hydroxy-3-buten-1-yl)-adenine (N6-HB-dA), N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA), and 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-?HMHP-dA). We have developed synthetic methodologies to incorporate site- and stereospecific N6-HB-dA, N6,N6-DHB-dA, 1,N6-?HMHP-dA adducts into DNA strands and examined their influence on DNA structure and replication. We found that N6-HB-dA did not distort DNA structure and was bypassed in an error-free manner by human lesion bypass polymerases ?, ?, ? and human polymerase �. In contrast, polymerase bypass of 1,N6-?HMHP-dA induced large numbers of A ? T and A ? C transversions and small deletions. Translesion synthesis across N6,N6-DHB-dA adducts by TLS polymerases was extremely inefficient. Steady-state kinetic experiments revealed that dTMP, dAMP, dCMP were incorporated with similar frequencies opposite N6,N6-DHB-dA lesion by TLS polymerases ? and ?. Sturctural NMR and thermal melting studies revealed that while regular Watson-Crick base pairing was conserved between N6-HB-dA and dT, N6,N6-DHB-dA and 1,N6-?HMHP-dA adducts destabilize the duplex by disrupting Watson-Crick hydrogen bonding. In summary, our results indicate that among BD-induced adenine adducts, 1,N6-?HMHP-dA is the most distorting and mutagenic, while N6,N6-DHB-dA is inefficiently bypassed by TLS polymerases.                                                

Translesion synthesis and structural studies of 1,3-butadiene-induced deoxyadenosine adducts

246th National Meeting (2013)

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