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

大肠杆菌中 DNA-蛋白质交联的处理和后果

基本信息

批准号：
7995717
负责人：
KENNETH N KREUZER
金额：
$ 3.79万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-20 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7995717
关键词：
Affect Aftercare Anti-Bacterial Agents Antibiotics Antineoplastic Agents Azacitidine Bacterial DNA Binding Biochemical Biological Models Cell Extracts Cells Chemicals Chemotherapy-Oncologic Procedure Chromosomes, Human, Pair 5 Ciprofloxacin Collection Complex Coupled Cytosine DNA DNA Damage DNA Gyrase DNA Methyltransferase DNA Modification Methylases DNA Polymerase III DNA Repair DNA Sequence DNA-Binding Proteins DNA-Directed RNA Polymerase DNA-protein crosslink Defect Deoxycytidine Enzymes Escherichia coli Exonuclease Failure Formaldehyde Genes Genetic Transcription Genomic Instability Goals Hand Hypersensitivity In Vitro Lead Lesion Malignant Neoplasms Measures Methyltransferase Modeling Nature Oligonucleotides Pathway interactions Pharmaceutical Preparations Phosphotyrosine Prevalence Process Proteins Public Health Quality Control Quinolone Antibiotic Quinolones RNA Radiation Reaction Research Ribosomes Role Scientist Site Syndrome System Testing Training Translating Translations Work analog base chemotherapeutic agent crosslink cytotoxicity design follow-up genetic analysis helicase improved in vivo inhibitor/antagonist interest leukemia methyl group mutant nuclease public health relevance repaired research study response tmRNA

Affect Aftercare Anti-Bacterial Agents Antibiotics Antineoplastic Agents Azacitidine Bacterial DNA Binding Biochemical Biological Models Cell Extracts Cells Chemicals Chemotherapy-Oncologic Procedure Chromosomes, Human, Pair 5 Ciprofloxacin Collection Complex Coupled Cytosine DNA DNA Damage DNA Gyrase DNA Methyltransferase DNA Modification Methylases DNA Polymerase III DNA Repair DNA Sequence DNA-Binding Proteins DNA-Directed RNA Polymerase DNA-protein crosslink Defect Deoxycytidine Enzymes Escherichia coli Exonuclease Failure Formaldehyde Genes Genetic Transcription Genomic Instability Goals Hand Hypersensitivity In Vitro Lead Lesion Malignant Neoplasms Measures Methyltransferase Modeling Nature Oligonucleotides Pathway interactions Pharmaceutical Preparations Phosphotyrosine Prevalence Process Proteins Public Health Quality Control Quinolone Antibiotic Quinolones RNA Radiation Reaction Research Ribosomes Role Scientist Site Syndrome System Testing Training Translating Translations Work analog base chemotherapeutic agent crosslink cytotoxicity design follow-up genetic analysis helicase improved in vivo inhibitor/antagonist interest leukemia methyl group mutant nuclease public health relevance repaired research study response tmRNA

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项目摘要

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.

描述（由申请人提供）：该项目的广泛远程目标是了解两种主要类别的化学治疗剂如何引起DNA损伤以及细胞如何处理这些药物诱导的DNA-蛋白交联。尽管许多化学治疗剂以及辐射和其他化学物质的流行率很高，但DNA-蛋白交联的修复知之甚少。靶向细菌DNA陀螺酶的奎诺酮抗生素稳定了一个中间体，在该反应中，酶通过磷酸酪氨酸键共价连接到断裂的DNA分子上。另一方面，抗癌药物5-氮杂丁胺在酶的识别位点导致胞嘧啶甲基转移酶和DNA之间的共价复合物，在该酶的识别位点没有DNA断裂。我们已经表明，两类抑制剂导致在酶共价结合到DNA的位点的复制叉上。第一个目的旨在阐明用喹诺酮治疗后DNA断裂和细胞毒性的机制，重点是多种基因的作用，包括DNAQ，RECQ，XSEAB和RUVAB。第二个目的分析了对5-氮杂丁胺过敏的突变体，该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许多实验测试进行探测，主要目标是确定受影响从DNA蛋白质蛋白交叉链接修复DNA损伤的突变体的子集。第三目的使用生化方法研究两类化学治疗剂形成的DNA-蛋白交联的修复和加工。这包括对体内形成的中间体的生化分析，以及在体外实验中，我们分析了与细胞提取物或纯化蛋白质反应中DNA-蛋白交联的命运。最后，在第四个目标中，我们跟进了最近的观察结果，即TMRNA转化质量控制系统对于5-余丁胺治疗后的生存至关重要。我们已经提出，DNA-蛋白交联块转录，从而导致耦合翻译核糖体的阻塞，以触发TMRNA系统的作用。将测试该模型的各个方面，包括位点特异性交联在触发TMRNA系统中的作用以及在阻塞的复合物中RNA和RNA聚合酶的命运。这项研究与公共卫生相关性与公共卫生有关，因为了解化学治疗剂的工作如何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学治疗剂：喹诺酮类药物是最常开处方的抗生素（包括常用的环丙沙星）和5-偶氮丁烷，可有效地抗白血病和美律综合症。此外，复制叉失效的途径和DNA蛋白交联的修复是与基因组不稳定性有关的问题，可能导致癌症的形成。