Base Analog Toxicity and Detoxification

描述（由申请人提供）：碱类似物作为有效抗病毒药、抗微生物剂和抗癌剂的用途受到其对宿主生物体的毒性的限制。这种毒性归因于通过抑制关键酶或超诱变而使常规核苷酸代谢中毒。我们最近证明，即使是天然碱基类似物尿嘧啶和次黄嘌呤，这是不知道引起任何上述后果，仍然是遗传毒性的大肠杆菌。因为它们诱导染色体断裂。事实证明，染色体片段化是核苷酸库不平衡和非规范DNA前体污染的重要后果，尽管经常被忽视。我们建议研究大肠杆菌rdgB、dut、tdk和thyA突变体中核苷酸池不平衡和污染的染色体后果。1）核苷酸池中修饰的DNA前体和DNA中修饰的碱基是什么？2)修饰的DNA前体合成和修饰的碱基修复的途径是什么？3)染色体断裂的机制是什么？是由修饰的碱基结合到DNA中引起的？为此，我们将使用以下方法：二维薄层色谱法检测修饰的DNA前体，酶切并随后进行后标记以识别DNA中修饰的核苷酸;分离与rdgB、tdk和dut基因合成致死的突变体，以揭示看似不相关的代谢途径之间的因果纠正相互作用;分离具有rdgB、tdk和dut失活的合成致死性的抑制子，以揭示合成致死性背后的机制;测定rdgB、tdk和dut突变体的突变谱;脉冲场凝胶电泳研究这些突变体的染色体断裂机制。拟议的研究将导致更好地了解碱基类似物的致染色体断裂潜力，以及阐明细胞的染色体断裂避免策略。

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.