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DnaA蛋白是广宿主范围质粒RK2在大肠杆菌中最小复制子复制所必需的。

DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli.

作者信息

Gaylo P J, Turjman N, Bastia D

机构信息

Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.

出版信息

J Bacteriol. 1987 Oct;169(10):4703-9. doi: 10.1128/jb.169.10.4703-4709.1987.

DOI:10.1128/jb.169.10.4703-4709.1987
PMID:2820940
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC213843/
Abstract

The minimal origin of replication of the broad-host-range plasmid RK2 has two potential recognition sequences for the DnaA protein of Escherichia coli. DNA transfer by transformation into a dnaA-null mutant of E. coli showed that DnaA protein is needed for replication or maintenance of mini-RK2. We isolated and purified DnaA protein as a chimeric protein, covalently attached to a piece of collagen and beta-galactosidase. The hybrid protein specifically bound to restriction fragments from the oriV region of RK2, which contained the two dnaA boxes. Deletion of the second dnaA box inactivated the origin and abolished the binding of the hybrid protein to the DNA fragment that had suffered the deletion. When the second dnaA box was replaced with an EcoRI linker of identical length, origin activity was restored. Binding experiments showed that the linker provided a weak dnaA box. An alternative explanation was that the linker restored proper spacing between sequences on either side of the deleted box, thus restoring origin activity.

摘要

广宿主范围质粒RK2的最小复制起点有两个潜在的大肠杆菌DnaA蛋白识别序列。通过转化导入大肠杆菌的dnaA基因缺失突变体进行DNA转移实验表明，DnaA蛋白对于mini-RK2的复制或维持是必需的。我们分离并纯化了作为嵌合蛋白的DnaA蛋白，该蛋白与一段胶原蛋白和β-半乳糖苷酶共价连接。这种杂交蛋白特异性地结合到RK2 oriV区域的限制性片段上，该区域包含两个dnaA框。删除第二个dnaA框会使起点失活，并消除杂交蛋白与发生缺失的DNA片段的结合。当第二个dnaA框被等长的EcoRI接头取代时，起点活性得以恢复。结合实验表明，该接头提供了一个弱dnaA框。另一种解释是，该接头恢复了缺失框两侧序列之间的适当间距，从而恢复了起点活性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/58754a23b7f7/jbacter00200-0302-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/b7ef0432b072/jbacter00200-0298-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/0d22cffc1b8b/jbacter00200-0300-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/0d9b3dcaf603/jbacter00200-0301-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/58754a23b7f7/jbacter00200-0302-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/b7ef0432b072/jbacter00200-0298-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/0d22cffc1b8b/jbacter00200-0300-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/0d9b3dcaf603/jbacter00200-0301-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/09d4/213843/58754a23b7f7/jbacter00200-0302-a.jpg

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本文引用的文献

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Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells.广宿主范围质粒RK2编码多个对大肠杆菌宿主细胞有潜在致死性的kil基因。
Proc Natl Acad Sci U S A. 1982 Mar;79(6):1935-9. doi: 10.1073/pnas.79.6.1935.
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Primary structure of the essential replicon of the plasmid pSC101.质粒pSC101必需复制子的一级结构。
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3
Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2.广宿主范围质粒RK2的trfB和korB区域的分子遗传学分析。
J Bacteriol. 2004 Jun;186(12):3785-93. doi: 10.1128/JB.186.12.3785-3793.2004.
4
Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis.酵母正向和反向双杂交分析揭示的DnaA-RepA相互作用的机制方面
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Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101.DnaB解旋酶被招募至质粒pSC101复制起点的机制。
Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):73-8. doi: 10.1073/pnas.96.1.73.
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Mechanistic studies of initiator-initiator interaction and replication initiation.引发剂-引发剂相互作用与复制起始的机制研究
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The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone.广宿主质粒RK2的复制起始蛋白由ClpX伴侣蛋白激活。
Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14378-82. doi: 10.1073/pnas.94.26.14378.
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Characterization of the replicon of plasmid pSW500 of Erwinia stewartii.斯氏欧文氏菌质粒pSW500复制子的特性分析
Mol Gen Genet. 1996 Apr 10;250(6):699-704. doi: 10.1007/BF02172981.
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Characterization of the basic replicon of pCM1, a narrow-host-range plasmid from the moderate halophile Chromohalobacter marismortui.嗜盐嗜碱菌死海嗜盐杆菌来源的窄宿主范围质粒pCM1基本复制子的特性分析
J Bacteriol. 1995 Jun;177(12):3443-50. doi: 10.1128/jb.177.12.3443-3450.1995.
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Regulation of cellular differentiation in Caulobacter crescentus.新月柄杆菌中细胞分化的调控
Microbiol Rev. 1995 Mar;59(1):31-47. doi: 10.1128/mr.59.1.31-47.1995.
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J Mol Biol. 1984 May 25;175(3):251-62. doi: 10.1016/0022-2836(84)90347-4.
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Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence.来自海洋细菌哈维氏弧菌的染色体复制起点在大肠杆菌中发挥作用：oriC共有序列。
Proc Natl Acad Sci U S A. 1983 Mar;80(5):1164-8. doi: 10.1073/pnas.80.5.1164.
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The locus of sequence-directed and protein-induced DNA bending.序列导向和蛋白质诱导的DNA弯曲位点。
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Molecular cloning and mapping of SphI restriction fragments of plasmid RP4.质粒RP4的SphI限制性片段的分子克隆与定位
Plasmid. 1983 Nov;10(3):303-7. doi: 10.1016/0147-619x(83)90047-1.
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Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA.大肠杆菌中质粒RK2的必需基因：trfB区域控制着trfA附近的一个kil基因。
J Bacteriol. 1983 Nov;156(2):584-91. doi: 10.1128/jb.156.2.584-591.1983.
9
Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication.纯化的DnaA蛋白在大肠杆菌染色体复制起点处启动复制过程中的作用。
Proc Natl Acad Sci U S A. 1983 Oct;80(19):5817-21. doi: 10.1073/pnas.80.19.5817.
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The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding.用β-半乳糖苷酶标记的质粒R6K的复制起始蛋白表现出序列特异性DNA结合。
Cell. 1983 Jan;32(1):131-40. doi: 10.1016/0092-8674(83)90503-2.