Base Analog Toxicity and Detoxification

碱基类似物作为有效的抗病毒剂、抗微生物剂和抗癌剂的用途受到它们的限制。 对宿主生物体的毒性。这种毒性归因于常规核苷酸的中毒 通过抑制关键酶或超诱变来代谢。我们最近证明，即使 天然碱基类似物尿嘧啶和次黄嘌呤，它们不知道会引起任何上述结果， 在E.因为它们诱导染色体断裂。结果是染色体 片段化是核苷酸库不平衡的重要后果，尽管常常被忽视， 非规范DM前体的污染。我们建议研究染色体 在大肠杆菌的rdgB、dut、tdk和thyA突变体中核苷酸库不平衡和污染的后果。 1）核苷酸池中修饰的DMA前体是什么， DNA中的修饰碱基2)修饰的DMA前体合成和修饰的途径是什么？ 基地修复？3)染色体断裂的机制是什么？ 改造成DNA为此，我们将采用以下方法：二维薄层 层析以检测修饰的DNA前体，酶切，随后进行后标记， 鉴定DNA中修饰的核苷酸;分离具有rdgB、tdk和dut基因的合成致死突变体， 揭示看似不相关的代谢途径之间的因果校正相互作用; 用rdgB、tdk和dut失活分离合成致死性抑制因子，以揭示其机制 背后的合成致死性; rdgB，tdk和dut突变体的突变谱的测定;脉冲- 场凝胶电泳研究这些突变体的染色体断裂机制。的 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。

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 DMAprecursors. 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 DMA precursors in the nucleotide pools and modified bases in DNA? 2) what are the pathways of the modified DMA 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.