REPLICATION OF CHLOROPLAST DNA

叶绿体 DNA 的复制

• 批准号: 3283677
• 负责人: KRISHNA K TEWARI
• 金额: $ 23.17万
• 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 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283677
• 关键词: DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; DNA topoisomerases; aminoacid analyzer; chloroplasts; circular DNA; electron microscopy; enzyme mechanism; enzyme structure; gene expression; genetic translation; laboratory rabbit; nucleic acid hybridization; nucleic acid sequence; plant genetics; proteins; radiotracer; tissue /cell culture

The long term goal of the proposal is to identify and purify the proteins involved in the replication of chloroplast DNA (ctDNA) followed by studying in vitro replication using purified proteins. Replication of ctDNA proceeds by the D-loop mechanism and involves both replicative intermediates produced by Cairn's mode of replication and rolling circle mechanism. We have mapped the origins of replication on the restriction endonuclease map of the pea ctDNA by electron microscopy. We have also developed a crude replication system that contains about 30 polypeptides and shows maximum DNA synthesis with recombinants pCP 12-7 and pCB 1-12 that have been found to contain the two D-loops. We now propose to progressively subclone these ctDNA fragments to eventually obtain the minimal ctDNA sequences that can replicate in an in vitro replication system. The exact sequences of the origins will be identified by obtaining deletion mutants followed by base substitution experiments. The D-loop DNA from supercoiled ctDNA will be analyzed for precisely mapping the initiation sites of replication and for the presence or absence of ribonucleotides as primers. We have obtained a pure preparation of topoisomerase I from the replication complex, and now plan to study its role in initiation of replication. We have obtained four DNA binding proteins whose role(s) in replication will also be studied. Experiments are proposed to find out whether a specific DNA primase or the chloroplast RNA polymerase serves to prime the initiation of replication. The replication of D-loop containing plasmids in vitro will be studied using pure DNA polymerase, topoisomerase I priming enzyme, DNA binding proteins, and any as yet unidentified proteins that are needed in the replication of ctDNA.

该提案的长期目标是识别和净化 参与叶绿体DNA复制的蛋白质 随后使用纯化的蛋白质研究体外复制。 ctDNA的复制通过D环机制进行，并涉及 这两种复制中间体都是由凯恩模式产生的， 复制和滚动循环机制。 我们绘制了 限制性内切酶图谱上的复制起点 电镜下观察到豌豆的ctDNA。 我们还开发了一种 复制系统，包含约30个多肽，并显示 最大的DNA合成与重组pCP 12-7和pCB 1-12， 都含有两个D环 我们现建议 逐步亚克隆这些ctDNA片段，以最终获得 可以在体外复制的最小ctDNA序列 复制系统 原点的确切序列将是 通过获得缺失突变体，然后是碱基 替代实验 超螺旋DNA中的D环DNA 将进行分析，以精确定位 复制和核糖核苷酸的存在或不存在， 引物 我们获得了拓扑异构酶I的纯制剂 从复制复杂，现在计划研究其作用， 复制的开始。 我们已经获得了四个DNA结合 还将研究在复制中起作用的蛋白质。 实验被提议找出是否一个特定的DNA引发酶 或者叶绿体RNA聚合酶用来启动 复制。 含D环质粒在大肠杆菌中的复制 体外研究将使用纯DNA聚合酶，拓扑异构酶I 引发酶，DNA结合蛋白，以及任何尚未鉴定的 ctDNA复制所需的蛋白质。

项目成果

Pea chloroplast DNA primase: characterization and role in initiation of replication.

豌豆叶绿体 DNA 引物酶：特征及其在复制起始中的作用。

• DOI: 10.1007/bf00016074
• 发表时间: 1991
• 期刊: Plant molecular biology
• 影响因子: 5.1
• 作者: Nielsen,BL;Rajasekhar,VK;Tewari,KK
• 通讯作者: Tewari,KK

Purification and properties of chloroplast DNA polymerase.

叶绿体 DNA 聚合酶的纯化和特性。

• DOI: 10.1016/0076-6879(86)18073-6
• 发表时间: 1986
• 期刊: Methods in enzymology
• 作者: Tewari,KK

Characterization of the pea chloroplast DNA OriA region.

豌豆叶绿体 DNA OriA 区域的表征。

• DOI: 10.1006/plas.1993.1052
• 发表时间: 1993
• 期刊: Plasmid
• 影响因子: 2.6
• 作者: Nielsen,BL;Lu,Z;Tewari,KK
• 通讯作者: Tewari,KK

Localization of replication origins in pea chloroplast DNA.

豌豆叶绿体 DNA 复制起点的定位。

• DOI: 10.1128/mcb.8.3.1216-1223.1988
• 发表时间: 1988
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Meeker,R;Nielsen,B;Tewari,KK
• 通讯作者: Tewari,KK

Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis.

• DOI: 10.1073/pnas.84.1.194
• 发表时间: 1987
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: B. Gold;N. Carrillo;K. Tewari;L. Bogorad