The action of 1-Nitroso-8-Nitropyrene in Escherichia Coli: DNA Adduct Formation and Mutational specificity in the laci Gene

Abstract

<p>1,8-Dinitropyrene (1, 8-DNP) is an environmental contaminant which is mutagenic and carcinogenic. In both prokaryotic and eukaryotic cells 1,8-DNP is metabolized by nitroreduction and O-acetylation to yield a nitrenium ion which binds to DNA. A metabolic intermediate is 1-nitroso-8-nitropyrene (1,8-NONP). This thesis examined the DNA adducts formed by 1,8-NONP, and the mutational specificity of 1,8-NONP in the lacI gene of E. coli. Three DNA adducts were formed in E. coli following treatment with 1,8-NONP. The major lesion (dG-C(8)-ANP), which comprised about 95% of the total adduct, was formed at the C(8) position of deoxyguanosine. It is likely that one of the minor adducts resulted from reaction with deoxyadenosine, while the other minor adduct was a product of either deoxyguanosine or deoxycytidine. The mutational specificity of 1,8-NONP was examined by cloning and sequencing several hundred lacI mutations which had been recovered from four E. coli strains which differed with respect to DNA repair background. The results suggested that 1,8-NONP induced many different types of mutations, the most prominent of which were base substitutions, frameshifts and deletions. Frameshift mutations were induced in all E. coli strains tested. However, the extent of frameshift induction was relatively low in E. coli strains proficient in nucleotide excision repair, suggesting that the premutagenic adduct(s) can be efficiently recognized and repaired by the uvrABC excinuclease. 1,8-NONP-induced frameshift mutations occurred primarily at sequences of contiguous guanine residues, and were characterized by the loss of G:C base pairs. The frameshift mutatians exhibited marked site specificity, consistent with an "incorporation-slippage" model for frameshift mutation. In some cases inverted and direct repeats might contribute to frameshift mutagenesis. The vast majority of frameshift mutations occurred at G:C base pairs, and were probably targeted by the major dG-C(8)-ANP adduct. Base substitution mutation was strongly influenced by cellular error-prone repair functions. Most of the base substitutions were G:C => T:A transversions and were probably targeted by the major dG-C(8)-ANP adduct. However, a small number of A:T => T:A transversions might have been targeted by the minor deoxyadenosine adduct. The nature of the base substitutions induced by 1,8-NONP suggests that adenine is the most comnon base incorporated during error-prone bypass of the bulky DNA adducts. The endpoints of most deletion mutations were G:C rich and contained direct repeats, consistent with a slippage model for deletion mutation. Some deletion endpoints contained sequences which were similar to the putative recognition sequences for enzymes which nick DNA during replication and repair.</p>