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Molecular Mechanism for Initiation of DNA Replication of ColE2 and Co1E3 Plasmids

ColE2 和 Co1E3 质粒 DNA 复制启动的分子机制

基本信息

批准号：
63480510
负责人：
ITOH Tateo
金额：
$ 4.35万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480510/
关键词：
Colicin E2 Colicin E3 Replication initiator protein Replication origin Plasmid-specificity Primer RNA Primase Reconstituted system 一方向複製プライマーRNA

项目摘要

The basic replicons of ColE2 and ColE3 consist of three functional portions : A gene for the plasmid-specific initiator protein (Rep ; 33-35 Kda) ; an origin (32 and 33 bp) capable of initiating replication in the presence of the Rep protein ; a gene for an antigens RNA (RNA I ; 115 n) complementary to the 5' untranslated region of the Rep mRNA and negatively regulating initiation of replication.Specificity of interaction of the Rep proteins and the origins is determined by the 20 amino acid-region near the carboxy terminal portion of the Rep protein of each plasmid and a single base pair deletion (ColE2) or insertion (ColE3) in the origin.The partially purified ColE2 Rep protein was shown to bind specifically to the ColE2 origin by using the filter-binding method.In vitro replication of ColE2 and ColE3 DNA in crude E. Coli cell extracts requires the Rep proteins and host DNA polymerase I but not RNA polymerase and DnaG primase. Replication starts from a region containing the cloned origin and proceeds unidirectionally. The 5' end of the newly synthesized leading strand was localized at a fixed position within the origin. The 3' end of the newly synthesized lagging strand was localized at the downstream end of the origin. This seems to be the basic mechanism for the unidirectional replication. The newly synthesized leading strand contains three ribonucleotide residues (ppApGpA) at its 5' end.The ColE2 Rep protein in conjunction with the host DNA polymerase I and single-strand DNA binding protein specifically starts the leading strand synthesis at the identical position with the identical primer RNA. The reaction specifically requires ADP in addition to ATP and GTP and incorporates ADP into the primer RNA. The Rep protein seems to be a novel sequence-specific primase capable of functioning on double-strand DNA.

ColE2和ColE3的基本复制子由三个功能部分组成：质粒特异性启动蛋白基因（Rep; 33-35 Kda）；一个在Rep蛋白存在下能够启动复制的起始点（32和33 bp）；一种抗原RNA （RNA I; 115 n）的基因，与Rep mRNA的5'非翻译区互补，负向调节复制的起始。Rep蛋白与原点相互作用的特异性取决于每个质粒中Rep蛋白羧基末端附近的20个氨基酸区域以及原点中单个碱基对的缺失（ColE2）或插入（ColE3）。部分纯化的ColE2 Rep蛋白通过过滤结合方法特异性结合ColE2起源。ColE2和ColE3 DNA在大肠杆菌粗提取液中的体外复制需要Rep蛋白和宿主DNA聚合酶I，而不需要RNA聚合酶和DnaG引物酶。复制从包含克隆源的区域开始，单向进行。新合成的前导链的5'端定位在原点内的固定位置。新合成的滞后链的3'端定位在起源的下游端。这似乎是单向复制的基本机制。新合成的前导链在其5'端含有三个核糖核苷酸残基（ppApGpA）。ColE2 Rep蛋白与宿主DNA聚合酶I和单链DNA结合蛋白结合，在具有相同引物RNA的相同位置特异性地开始前导链合成。除了ATP和GTP外，该反应还需要ADP，并将ADP纳入引物RNA中。Rep蛋白似乎是一种新的序列特异性引物酶，能够在双链DNA上起作用。