FUNCTIONAL CONSEQUENCES OF 2-CHLOROADENINE IN DNA

DNA 中 2-氯腺嘌呤的功能后果

• 批准号: 3199955
• 负责人: Patricia Hentosh
• 金额: $ 13.57万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-05 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3199955
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; RNA biosynthesis; adenosine triphosphate; antineoplastics; bacteriophage phi X174; chemical substitution; deoxyadenosines; exonuclease; gel mobility shift assay; gene mutation; genetic promoter element; laboratory mouse; ligase; nucleic acid sequence; site directed mutagenesis; transfection

2-Chloro-2'-deoxyadenosine, a nucleoside analog with potential efficacy in the treatment of lymphoid neoplasias, is under active investigation in Phase II pediatric and adult clinical trials. The compound's ultimate clinical value will depend on a more complete understanding of its mechanism(s) of action, its mutagenicity, and its repairability upon incorporation into DNA, which could be a factor in therapeutic efficacy. Hence, the overall goal of this project is to determine the functional consequences of incorporation of the analog's triphosphate form, CldATP, into DNA. This event is postulated to interfere with subsequent DNA synthesis and RNA transcription, to alter the fidelity of DNA polymerization, and to be recognized as an abnormal base by proteins involved in DNA repair. These predictions will be tested in four series of experiments designed with the following aims: Specific Aim 1. Determine the effects of CldATP incorporation on subsequent DNA polymerization by examining the susceptibility of ClA-containing nascent DNA strands to 3'-5' exonucleolytic degradation, the efficiency of bond formation between ClA residues and adjacent nucleotides, and the ability of DNA-containing ClA residues to serve as a template for in vitro DNA synthesis. Specific Aim 2. Determine the fidelity of DNA polymerization in the presence of ClA by site-directed mutagenesis and the phiX174am16 assay that detects single base substitutions within an amber mutation, by use of the M13mp2 forward mutation assay that detects changes at many sites within a nonessential gene, and by the analysis of mammalian cell mutation induction within the human hgprt locus and in a transfected phage shuttle vector. Specific Aim 3. Determine the effects of ClA-substituted DNA on RNA transcription by examining promoter function, the extent and rate of elongation of RNA transcripts, and fidelity of base pairing during in vitro transcription of ClA-containing DNA. Specific Aim 4. Identify DNA-binding proteins or DNA repair enzymes specific for ClA residues by first assaying for recognition of this abnormal base in DNA by purified E. coli ABC excinuclease and, secondly, by examining human cell extracts in a gel shift binding assay to identify novel DNA binding proteins that bind to or repair ClA-containing DNA. The information gained from these studies will provide needed insights into the effects, both short- and long-term, of a chlorinated purine in DNA. In addition, it may be possible from the mutagenicity data to predict the analog's carcinogenic potential or lack thereof.

2-氯-2'-脱氧腺苷，具有潜在功效的核苷类似物 在治疗淋巴性肿瘤时，正在积极研究 II期小儿和成人临床试验。 该化合物的终极 临床价值将取决于对其的完整理解 动作的机制，其诱变性及其可修复性 纳入DNA，这可能是治疗功效的一个因素。 因此，该项目的总体目标是确定功能 掺入类似物三磷酸形式的后果，cldatp， 进入DNA。 假定此事件会干扰随后的DNA 合成和RNA转录，以改变DNA的保真度 聚合，并被蛋白质识别为异常碱 参与DNA修复。 这些预测将在四个系列中进行测试 具有以下目的设计的实验：特定目标1。 确定CLDATP掺入对随后DNA的影响 通过检查含CLA的新生的敏感性来聚合 DNA链至3'-5'外核解水解降解，粘结效率 CLA残基和相邻核苷酸之间的形成，能力 含DNA的CLA残基作为体外DNA的模板 合成。 具体目标2。确定DNA聚合的保真度 在通过位置定向诱变和phix174AM16的CLA存在下 检测琥珀突变中单碱基取代的测定，通过 使用M13MP2正向突变测定法，该测定在许多情况下检测到变化 非必需基因内的位点，并通过哺乳动物细胞的分析 人类HGPRT基因座和转染中的突变诱导 噬菌体穿梭矢量。 特定目标3。确定 通过检查启动子功能，在RNA转录上取代的DNA， RNA转录本的伸长程度和速率和忠诚度 在含CLA的DNA的体外转录过程中碱基配对。 特定目标4。识别结合蛋白或DNA修复酶 首先要识别CLA残留物的特定于CLA残留 通过纯化的大肠杆菌ABC精核酸酶，在DNA中的异常碱，其次， 通过检查凝胶转移结合测定中的人类细胞提取物以识别 与结合或修复含CLA的DNA结合或修复的新型DNA结合蛋白。 从这些研究中获得的信息将提供所需的见解 在短期和长期的氯化嘌呤中的影响 脱氧核糖核酸。 另外，从诱变性数据到 预测模拟的致癌潜力或缺乏。