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Sequence context modulation of polycyclic aromatic hydrocarbon‐induced mutagenesis
Author(s) -
Chary Parvathi,
Stone Michael P.,
Lloyd R. Stephen
Publication year - 2013
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.21806
Subject(s) - klenow fragment , mutagenesis , context (archaeology) , transversion , chemistry , stereochemistry , adduct , mutation , benzo(a)pyrene , microbiology and biotechnology , exonuclease , dna , genetics , biology , biochemistry , carcinogen , dna polymerase , gene , paleontology , organic chemistry
DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[ a ]pyrene‐7,8‐dihydrodiol‐9,10‐epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N 6 ‐BPDE dA adducts at the second position in the N‐ ras codon 61(CAA) (61 2 ) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C 10 S ‐BPDE and C 10 R ‐BPDE lesions linked through the N 6 position of adenine in the sequence context N‐ ras codon 61, position 3 (C 10 S ‐BPDE and C 10 R ‐BPDE at 61 3 ) was carried out in Escherichia coli , and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C 10 S ‐BPDE and C 10 R ‐BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease‐deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N‐ ras 61 3 resulted in truncated products, with the C 10 S ‐BPDE adducts being more blocking than C 10 R ‐BPDE lesions, and termination of synthesis was more pronounced at position 61 3 than at 61 2 for each of the lesions. Environ. Mol. Mutagen. 54:652–658, 2013. © 2013 Wiley Periodicals, Inc.