Base Analog Toxicity and Detoxification

说明(申请人提供)：碱基类似物作为有效的抗病毒药物、抗菌剂和抗癌剂的使用受到它们对宿主生物体的毒性的限制。这种毒性可归因于通过抑制关键酶而毒化正常的核苷酸新陈代谢或高度诱变。我们最近已经证明，即使是已知不会引起上述任何后果的天然碱基类似物尿嘧啶和次黄嘌呤，在大肠杆菌中仍然具有遗传毒性，因为它们会导致染色体碎裂。事实证明，染色体断裂是核苷酸池失衡和非规范DNA前体污染的一个重要的、但经常被忽视的后果。我们建议在以下问题的指导下，研究核苷酸池失衡和污染对大肠杆菌rdgB、dut、Tdk和Thya突变体的染色体后果：1)核苷酸池中的修饰DNA前体和DNA中的修饰碱基是什么？2)修饰DNA前体合成和修饰碱基修复的途径是什么？3)DNA中加入修饰碱基导致染色体断裂的机制是什么？为此，我们将使用以下方法：二维薄层层析检测修饰的DNA前体，酶切后标记鉴定DNA中的修饰核苷酸；分离与rdgB、Tdk和dut基因合成致死的突变体，以揭示看似不连锁的代谢途径之间的因果纠正相互作用；分离具有rdgB、tdk和dut失活的合成致死抑制物，以揭示合成致死背后的机制；测定rdgB、tdk和dut突变体的突变谱；脉冲场凝胶电泳法研究这些突变体的染色体断裂机制。这项拟议的研究将有助于更好地理解碱基类似物的断裂潜能，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.