Base Analog Toxicity and Detoxification

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DESCRIPTION (provided by applicant): The use of base analogs as effective antivirals, antimicrobials and anti-cancer agents is limited by their toxicity to the host organism. This toxicity is attributed to either poisoning of the regular nucleotide metabolism by inhibiting key enzymes or to hypermutagenesis. We have recently demonstrated that even natural base analogs uracil and hypoxanthine, that are not known to elicit any of the above consequences, are still genotoxic in E. coli because they induce chromosomal fragmentation. It turns out that chromosomal fragmentation is an important, though often ignored, consequence of the nucleotide pool imbalance and contamination with non-canonical DNA precursors. We propose to investigate the chromosomal consequences of nucleotide pool imbalance and contamination in the rdgB, dut, tdk and thyA mutants of E. coli guided by the following questions: 1) what are the modified DNA precursors in the nucleotide pools and modified bases in DNA? 2) What are the pathways of the modified DNA precursor synthesis and modified base repair? 3) What are the mechanisms of chromosomal fragmentation, caused by incorporation of the modified bases into DNA? To this end, we will use the following methods: 2-dimensional thin layer chromatography to detect modified DNA precursors, enzymatic excision with subsequent post-labeling to identify modified nucleotides in DNA; isolation of mutants synthetic lethal with the rdgB, tdk and dut genes to reveal cause-consequence-correction interactions between seemingly unlinked metabolic pathways; isolation of suppressors of synthetic lethalities with rdgB, tdk and dut inactivations to reveal the mechanisms behind the synthetic lethalities; determination of mutation spectra of the rdgB, tdk and dut mutants; pulsed-field gel electrophoresis to study mechanisms of the chromosomal fragmentation in these mutants. The proposed research will lead to a better understanding of the clastogenic potential of base analogs, as well as to elucidation of the chromosomal breakage-avoidance strategies of the cell.