REPLICATION OF CHLOROPLAST DNA

叶绿体 DNA 的复制

• 批准号: 3283672
• 负责人: KRISHNA K TEWARI
• 金额: $ 7.52万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 1987-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283672
• 关键词: DNA directed DNA polymerase; DNA topoisomerases; aminoacid analyzer; chloroplasts; circular DNA; electron microscopy; enzyme structure; gene expression; genetic translation; nucleic acid sequence; plant population genetics; radiotracer; tissue /cell culture

The long term goal of this proposal is to establish a detailed in vitro replication system for chloroplast (ct) DNA. Replication of ctDNA proceeds by the D-loop mechanism and involves both Cairn's replicative intermediates and those replicative intermediates produced by the rollig circle mechanism. A replication transcription complex (RTC) from chloroplasts that consists of 30-50 polypeptides has been isolated. This complex can be obtained either with endogenous ctDNA (DNA-RTC comlex) or without the DNA (RTC complex). A DNA polymerase, a DNA binding protein, and an RNA polymerase were purified from this complex. The first phase of research will concentrate on initiation of ctDNA replication and elongation of the newly synthesized DNA chains. Sites of initiation of replication will be identified on the restriction endonuclease map of pea ctDNA by cross linking the supercoiled DNA with trioxsalene, followed by restriction and electron microscopic analysis of the DNA fragments containing D-loops. Precise position of D-loops will be determined by in vitro 5 feet end labeling using polynucleotide kinase and Gamma-labeled dTTP, purifying the labeled D-loops in denaturing gels, hybridizing the D-loops with an appropriate restriction fragment of ctDNA, single strand nuclease digestion, and analysis in the DNA sequencing ladder. The in vitro replication process will be studies using RTC comlex and supercoiled DNA as a template. IN parallel with these studies, we will purify two more enzymes from the RTC complex. The first enzyme will be topoisomerase, whose presence in chloroplasts is already identified. The second enzyme will be DNA ligase. Using topoisomerase, DNA polymerase, DNA binding protein, and DNA ligase, we will attempt to reconstitute the system to see whether DNA synthesis takes place using supercoiled ctDNA. It is hoped that such a system will at least carry out extension of already formed D-loops because D-looped molecules resemble a gapped DNA molecule, which is the preferred substrate of the ctDNA polymerase.

本提案的长期目标是建立详细的体外 叶绿体DNA复制系统。 ctDNA的复制进行 通过D环机制，并涉及凯恩的复制中间体 而那些由旋转圈产生的复制中间体 机制 叶绿体复制转录复合体 由30 - 50个多肽组成的蛋白质。 这个复合体可以是 用内源性ctDNA（DNA-RTC复合物）或不用DNA获得 (RTC复合物）。 DNA聚合酶、DNA结合蛋白和RNA 从该复合物中纯化聚合酶。 第一阶段研究 将集中于ctDNA复制的起始和 新合成的DNA链 复制起始位点为 通过杂交在豌豆ctDNA的限制性内切酶图谱上鉴定 将超螺旋DNA与trioxsalene连接，然后进行限制， 含有D环的DNA片段的电子显微镜分析。 D环的精确位置将由体外5英尺末端确定 使用多核苷酸激酶和γ-标记的dTTP标记，纯化 在变性凝胶中标记D-环，将D-环与 合适的ctDNA限制性片段，单链核酸酶 消化和DNA测序梯中的分析。 体外 复制过程将使用RTC复合物和超螺旋DNA作为 模板。 在进行这些研究的同时，我们将再纯化两个 RTC复合物中的酶。 第一种酶是拓扑异构酶， 它在叶绿体中的存在已经被确认。 第二酶 DNA连接酶 使用拓扑异构酶、DNA聚合酶、DNA结合 蛋白质和DNA连接酶，我们将尝试重建系统， DNA合成是否使用超螺旋ctDNA进行。 希望 这样一个系统将至少执行已经形成的 D环，因为D环分子类似于有间隙的DNA分子， ctDNA聚合酶的优选底物。