Processing and consequences of DNA-protein crosslinks in E. coli

描述（由申请人提供）：本项目的长期目标是了解两大类化疗药物如何引起DNA损伤，以及细胞如何处理这些药物诱导的DNA-蛋白质交联。尽管dna -蛋白质交联在许多化疗药物、辐射和其他化学物质中普遍存在，但人们对它们的修复知之甚少。喹诺酮类抗生素以细菌DNA回转酶为目标，稳定了一种反应中间体，在这种反应中间体中，酶通过磷酸酪氨酸键共价地附着在断裂的DNA分子上。另一方面，抗癌药物5-氮杂胞苷在胞嘧啶甲基转移酶和DNA的识别位点上形成共价复合物，复合物中没有DNA断裂。我们已经证明，这两类抑制剂导致在酶与DNA共价结合的位点上的复制叉阻塞。第一个目的旨在阐明喹诺酮类药物治疗后DNA断裂和细胞毒性的机制，重点研究包括dnaQ、recQ、xseAB和ruvAB在内的几个基因的作用。第二个目的是分析对5-氮杂胞苷敏感的突变体，这种突变体诱导共价甲基转移酶- dna复合物。他们的超敏反应的基础将通过一些实验测试来探索，一个主要的目标是确定受DNA-蛋白质交联修复DNA损伤影响的突变体的子集。第三个目的是使用生化方法来研究由两类化疗药物形成的dna -蛋白质交联的修复和加工。这包括体内形成的中间体的生化分析，以及在体外实验中，我们分析dna -蛋白质交联与细胞提取物或纯化蛋白质反应的命运。最后，在第四个目标中，我们跟进了我们最近的观察，即tmRNA翻译质量控制系统对5-氮杂胞苷治疗后的生存很重要。我们提出dna -蛋白质交联阻断转录，导致耦合翻译核糖体的阻断，从而触发tmRNA系统的作用。该模型的各个方面将被测试，包括位点特异性交联在触发tmRNA系统中的作用，以及被阻断复合物中RNA和RNA聚合酶的命运。这项研究与公共卫生相关，因为了解化疗药物如何起作用可以帮助科学家和临床医生改善药物治疗。这里研究了两种重要的化疗药物：喹诺酮类药物，这是最常用的抗生素之一（包括常用的环丙沙星），以及5-阿扎胞苷，它在针对白血病和白血病前期综合征的癌症化疗中有效。此外，复制叉失效的途径和DNA-蛋白质交联的修复是与基因组不稳定相关的问题，可能导致癌症的形成。

DESCRIPTION (provided by applicant): The broad long-range goals of this project are to understand how two major classes of chemotherapeutic agents cause DNA damage and how cells deal with DNA-protein crosslinks which are induced by these agents. The repair of DNA-protein crosslinks is very poorly understood, in spite of their prevalence from many chemotherapeutic agents as well as radiation and other chemicals. The quinolone antibiotics, which target bacterial DNA gyrase, stabilize a reaction intermediate in which the enzyme is covalently attached to a broken DNA molecule via phosphotyrosine bonds. Anticancer drug 5-azacytidine, on the other hand, leads to covalent complexes between cytosine methyltransferase and DNA at the recognition sites of the enzyme, with no DNA break in the complex. We have shown that both classes of inhibitors lead to blockage of the replication fork at sites where the enzymes are covalently bound to DNA. The first aim seeks to elucidate the mechanism of DNA breakage and cytotoxicity after treatment with quinolones, with an emphasis on the role of several genes including dnaQ, recQ, xseAB, and ruvAB. The second aim analyzes mutants that are hypersensitive to 5-azacytidine, which induces covalent methyltransferase-DNA complexes. The basis of their hypersensitivity will be probed with a number of experimental tests, and a major goal is to identify a subset of the mutants affected for repair of DNA damage from the DNA-protein crosslinks. The third aim uses biochemical approaches to investigate the repair and processing of DNA-protein crosslinks formed by both classes of chemotherapeutic agents. This includes biochemical analyses of intermediates formed in vivo, as well as in vitro experiments where we analyze the fate of DNA-protein crosslinks in reactions with cell extracts or purified proteins. Finally, in the fourth aim, we follow up on our recent observation that the tmRNA translational quality control system is important for survival after 5-azacytidine treatment. We have proposed that the DNA-protein crosslinks block transcription, which leads to blockage of the coupled translating ribosomes, to trigger the tmRNA system into action. Aspects of this model will be tested, including the role of the site- specific crosslinks in triggering the tmRNA system and the fate of the RNA and RNA polymerase in the blocked complexes. PUBLIC HEALTH RELEVANCE This research is relevant to public health because understanding how chemotherapeutic agents work can help scientists and clinicians improve therapy with the drugs. Two important chemotherapeutic agents are studied here: the quinolones, which are among the most frequently prescribed antibiotics (including the commonly used ciprofloxacin), and 5- azacytidine, which is effective in cancer chemotherapy against leukemia and pre-leukemia syndromes. In addition, the pathways of replication fork failure and the repair of DNA- protein crosslinks are issues relevant to the genome instability that can lead to the formation of cancers.