Date of Completion

1-7-2020

Embargo Period

7-5-2020

Keywords

chemical carcinogens, 6-nitrochrysene, carcinogen-DNA adduct, mutagenesis, cancer

Major Advisor

Ashis K. Basu

Associate Advisor

Amy Howell

Associate Advisor

Alfredo Angeles-Boza

Associate Advisor

Mark Peczuh

Associate Advisor

Anwar Beshir

Field of Study

Chemistry

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The environmental pollutant, 6-nitrochrysene (6-NC) is the most potent carcinogen evaluated by the newborn mouse assay. The genotoxicity of 6-NC is derived from its ability to form electrophilic species in cells which can react with nucleophilic sites of 2'-deoxyguanosine (dG) and 2’-deoxyadenosine (dA) in DNA to generate DNA-carcinogen adducts. DNA lesions derived from 6-NC can play important roles in the development of human cancer by inducing mutations in crucial genes resulting in the disruption of gene expression. Mutations in an oncogene, a tumor-suppressor gene such as p53, or a gene that controls the cell cycle can lead to uncontrolled cell growth, resulting in carcinogenesis, a process which ultimately gives rise to human cancer. Most of these mutations arise through error-prone mutagenic bypass of the lesions which is enabled by low fidelity translesion synthesis (TLS) DNA polymerases.

6-NC is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields major DNA adducts at the C8 and N2 positions of dG, N-(dG-8-yl)-6-AC and 5-(dG-N2-yl)-6-AC. The nitroreduction pathway also yield adduct at the C8 position of dA, N-(dA-8-yl)-6-AC and a 2'-deoxyinosine (dI) adduct, N-(dI-8-yl)-6-AC; which is believed to be a product of deamination of the adenine adduct N-(dA-8-yl)-6-AC. The N-(dA-8-yl)-6-AC adduct is particularly interesting, a nucleotide excision repair assay demonstrated that it is repaired much more slowly than many other bulky DNA adducts, including other DNA adducts formed by 6-NC. Neither the total synthesis nor cellular replication properties of the N-(dA-8-yl)-6-AC or other adducts derived from 6-NC have ever been reported.

Chapter 2 describes synthetic methods developed to access 6-NC modified nucleosides, phosphoramidites and oligodeoxynucleotides of the C8-dA adduct, N-(dA-8-yl)-6-AC employing an optimized Buchwald-Hartwig palladium catalyzed cross-coupling strategy, which provided a high yield of the protected N-(dA-8-yl)-6-AC adducted nucleoside.The protected N-(dA-8-yl)-6-AC adduct was converted to the protected 3’-phosphoramidite monomer and site specifically incorporated into 12 and 15-mer oligodeoxynucleotides (ODNs) via automated solid-phase DNA synthesis. These oligodeoxynucleotides were purified by reverse phase HPLC followed by denaturing polyacrylamide gel electrophoresis and characterized by high resolution mass spectrometry.

Chapter 3 employs 2D NMR spectroscopy and DFT calculations to investigate the conformational preference of N-(dA-8-yl)-6-AC adducted nucleoside. We further investigated the helical structures of N-(dA-8-yl)-6-AC adduct in 12 and 15-mer oligodeoxynucleotide duplexes using circular dichroism (CD) spectroscopy. The stability of N-(dA-8-yl)-6AC adducted nucleoside was also investigated under biologically relevant conditions using UV/Vis spectroscopy and HPLC assays.

Chapter 4 describes investigation of the replication bypass efficiency of N-(dA-8-yl)-6-AC adduct in Escherichia coli and in human embryonic kidney (HEK293T) cells. The 15-mer containing N-(dA-8-yl)-6-AC modified oligodeoxynucleotide was used to construct single-stranded shuttle vectors containing a single N-(dA-8-yl)-6-AC adduct site using recombinant DNA technology. These recombinant vectors were replicated in Escherichia coli and HEK293T cells. The roles of several translesion synthesis (TLS) replication polymerases in adduct bypass was also investigated via conducting replication experiments in isogenic cells where individual TLS DNA polymerases were depleted by the CRISPR/Cas9 genome editing method or siRNA knockdown approach.

Chapter 5 evaluates the mutagenicity and replication bypass efficiency of the ring opened formamidopyrimidine(Fapy.dG) adduct that is produced via oxidative stress in two interesting p53 mutation hotspot sequences that include codons 248 and 249. Fapy.dG modified oligodeoxynucleotides was used to construct single-stranded shuttle vectors containing a single adducted site and replicated in HEK293T cells.

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