Influence of chemical structure of DNA lesion on their replication and repair in human cells

Exogenous agents like ionizing radiations and chemical carcinogens can attack the intracellular DNA and generate mutagenic DNA lesions which, unless repaired, may lead to genetic instability and cancer. This thesis which is divided into two distinct chapters focuses on a subset of DNA lesions derived from ionizing radiation and chemical carcinogens.;The first chapter of this thesis focuses on 8,5'-cyclo-2'-deoxynucleotides which are major tandem-lesions formed by ionizing radiation induced hydroxyl radical attack on 2'-deoxynucleosides. These lesions, which exist as 5' R- and 5'S-diastereomers are known to distort the DNA duplex and unlike other oxidative lesions, are repaired by nucleotide excision repair (NER) pathway. However, most of the biological repair studies done till date have been performed on 8,5'-cyclo-2'-deoxyadenosine (cdA) lesions whereas repair of 8,5'-cyclo-2'-deoxyguanosine (cdG) lesions is not known.;Therefore, in this thesis chapter, I have investigated the biological repair of S-cdG lesion and found that it gets repaired by NER in human cell extracts. When NER efficiency of S-cdG paired with different complementary bases was investigated, the duplex pair (S-cdG·T) that showed a departure from the normal Watson-Crick base pairing was excised more efficiently than duplex pair ( S-cdG·C) that maintained a near-normal Watson-Crick base pairing. Similar results were obtained for the repair of S-cdA containing DNA duplexes as well. Interestingly, the presence of deoxyadenosine opposite to both S-cdG and S-cdA lesions markedly reduced the efficiency of NER process suggesting that a complete lack of hydrogen bonding does not necessarily improve the repair. These results add to the complexity of human NER process and show that it discriminates the damaged base pairs on the basis of multiple criteria.;The second chapter of this thesis focuses on the mutagenicity of a potent heterocyclic aromatic amine (HAA), 2-amino-3-methylimidazolo[4,5- f]-quinoline (IQ) in human cells. HAAs were discovered in cooked meat about two decades ago but are still one of the most potent mutagens ever tested in Ames/Salmonella assay, with IQ turning out to be one of the most mutagenic members of this class. However, not much is known about the genotoxicity of IQ in humans, except that it gets bioactivated by human cytochrome P450 to generate two DNA adducts the C8- and N 2-dG-IQ.;Therefore, in this thesis chapter, I have investigated the genotoxicity of IQ mutagen in human cells by using C8- and N2-dG-IQ adducts which were site-specifically incorporated into G1, G 2 or G3 position of NarI sequence (5'-dCTC G1G2C G3CC ATC-3') that was shown to be a mutational hot-spot for DNA adducts formed by carcinogenic aromatic amines and HAAs. My results shows that both C8- and N2-dG-IQ adducts are mutagenic in human cells. For both adducts, G→T transversions were the major targeted mutations and C8-dG-IQ was found to be significantly more mutagenic at G3 position than either C8- or N 2-dG-IQ adduct placed at any other G sites in the NarI sequence. Currently, we are in the process of identifying DNA polymerase/s involved in replication past these IQ lesions and findings from this study will provide a much better insight into human health risk posed by these dietary carcinogens.