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长期目标是了解两个主要类别如何化疗药物引起 DNA 损伤以及细胞如何处理 DNA 蛋白由这些试剂诱导的交联。 DNA-蛋白质交联的修复非常有效尽管许多化疗药物也普遍存在这种现象，但对其了解甚少如辐射和其他化学物质。喹诺酮类抗生素，针对细菌 DNA 旋转酶，稳定反应中间体，其中酶共价连接到通过磷酸酪氨酸键断裂 DNA 分子。抗癌药物5-氮杂胞苷，另一方面，导致胞嘧啶甲基转移酶和 DNA 之间形成共价复合物酶的识别位点，复合物中没有 DNA 断裂。我们已经证明两类抑制剂都会导致酶所在位点的复制叉受阻与 DNA 共价结合。第一个目标是阐明 DNA 断裂和细胞毒性的机制用喹诺酮类药物治疗后，重点关注几个基因的作用，包括 dnaQ、recQ、xseAB 和 ruvAB。第二个目标是分析过敏突变体 5-氮杂胞苷，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一系列实验测试来探究，主要目标是鉴定受 DNA 蛋白修复 DNA 损伤影响的突变体子集交联。第三个目标使用生化方法来研究修复和由两类化疗药物形成的 DNA-蛋白质交联的处理。这包括体内和体外形成的中间体的生化分析我们分析 DNA-蛋白质交联在细胞反应中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，我们跟进最近的工作观察发现 tmRNA 翻译质量控制系统对于术后生存很重要 5-氮杂胞苷治疗。我们提出DNA-蛋白质交联阻断转录，导致偶联翻译核糖体的阻断，从而触发 tmRNA 系统发挥作用。该模型的各个方面将受到测试，包括站点的作用- 触发 tmRNA 系统的特定交联以及 RNA 和 RNA 的命运封闭复合物中的聚合酶。