Targeted A --> T and G --> T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol epoxide of dibenz[a,j]anthracene in M13mp7L2.
The studies described in this report directly examined the mutagenicity in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguanosine (dGuo) adduct derived from (+)-anti-dibenz[a,j]-anthracene-3,4-diol 1,2-epoxide [(+)anti-DB[a,j]A-DE] that were site-specifically placed in a single-stranded M13mp7L2 replication vector. An 11-base oligonucleotide (5'-CTC ACG CTT CT-3') containing either a single (+)anti-DB[a,j]A-DE--trans-N2-dGuo or (+)anti-DB[a,j]A-DE--trans-N6dAdo adduct was successfully incorporated into single-stranded M13mp7L2 plasmid via ligation. In vitro studies using E. coli DNA polymerase I (Klenow fragment)indicated that both adducts were effective blocks for polymerase action. E. coli strains JM103 and JM103 uvrA6 were subsequently transformed with control (unadducted) and adduct-containing M13mp7L2 constructs followed by analysis of progeny DNA. In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors. Activation of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacterial strain transformed with adduct-containing vectors. Targeted mutations were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors. In JM103 cells, (+)anti-DB[a,j]A-DE--N6-dAdo induced exclusively A --> t transversions and (+)anti-DB[a,j]A-DE--N2-dGuo induced exclusively G --> T transversions. In JM103 uvrA6 cells, similar targeted transversion mutations were also obtained except that a few C deletions (i.e., aprroximately 10% of the mutations) were detected immediately 3' to the dAdo adduct. While mutagenesis was SOS dependent in JM103 cells [<0.15% (-SOS) vs approximately 1.3% (+SOS)], it appeared to be SOS independent in JM103 uvrA6 cells (approximately 1-2% in the presence or absence of SOS induction). It is argued that adduct-induced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms. In contrast, A --> T mutations are unlikely to arise via a misinformational pathway, which provides the strongest support to date that bulky DNA adducts can induce mutations via a noninformational pathway.
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Science Park-Research Division, Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, USA.
Gill
R D
RD
Cortez
C
C
Harvey
R G
RG
Loechler
E L
EL
DiGiovanni
J
J
D000225:Adenine; D001483:Base Sequence; D001551:Benz(a)Anthracenes / Q000138:chemical synthesis / Q000494:pharmacology*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D003839:Deoxyadenosines; D003849:Deoxyguanosine; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006147:Guanine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000138:chemical synthesis / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017354:Point Mutation; D013941:Thymine; D014162:Transfection
D001551:Benz(a)Anthracenes; D018736:DNA Adducts; D004277:DNA, Single-Stranded; D003839:Deoxyadenosines; D009153:Mutagens; D009838:Oligodeoxyribonucleotides; C057927:dibenz(a,j)anthracene-3,4-diol-1,2-epoxide; D006147:Guanine; D003849:Deoxyguanosine; D000225:Adenine; D013941:Thymine