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：
8292095
负责人：
KENNETH N KREUZER
金额：
$ 32.49万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8292095
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 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 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 abstracting analog base chemotherapeutic agent crosslink cytotoxicity design follow-up genetic analysis helicase improved in vivo inhibitor/antagonist interest leukemia methyl group mutant nuclease repaired research study response tmRNA

项目摘要

Abstract 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.

抽象的该项目的广泛远程目标是了解两个主要类别化学治疗剂的导致DNA损伤以及细胞如何处理DNA蛋白这些药物诱导的交联。 DNA-蛋白交联的修复非常尽管许多化学治疗剂的流行率也很少了解作为辐射和其他化学物质。喹诺酮抗生素，靶向细菌DNA 回旋酶稳定反应中间体，在该中间体将酶共价附着在该酶通过磷酸酪氨酸键断裂的DNA分子。抗癌药5-氮杂丁胺，另一方面，导致胞嘧啶甲基转移酶和DNA之间的共价复合物在酶的识别位点，在复合物中没有DNA断裂。我们已经表明两类抑制剂都会导致酶的位点的复制叉阻塞共价结合到DNA。第一个目的旨在阐明DNA断裂和细胞毒性的机制用喹诺酮治疗后，重点是多种基因的作用 DNAQ，RECQ，XSEAB和RUVAB。第二个目标分析了过敏的突变体到5-氮杂丁胺，该丁胺诱导共价甲基转移酶-DNA复合物。的基础他们的超敏反应将通过许多实验测试进行探测，一个主要目标是确定因DNA-蛋白质修复DNA损伤的突变体的子集交联。第三目的使用生化方法来研究维修和由两类化学治疗剂形成的DNA-蛋白交联的处理。这包括对体内形成的中间体的生化分析以及体外我们分析与细胞反应中DNA-蛋白交联的命运的实验提取物或纯化的蛋白质。最后，在第四个目标中，我们跟进了最近的观察到TMRNA平移质量控制系统对于生存很重要 5-氮杂丁胺治疗。我们提出了DNA-蛋白交联块转录导致耦合翻译核糖体的阻塞，以触发 TMRNA系统采取行动。该模型的各个方面将进行测试，包括站点的作用 - 触发TMRNA系统和RNA和RNA的命运时的特定交联封闭的复合物中的聚合酶。