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RepA蛋白和DnaA蛋白是R1质粒体外复制起始所必需的，且与oriR序列相互作用。

RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence.

作者信息

Masai H, Arai K

出版信息

Proc Natl Acad Sci U S A. 1987 Jul;84(14):4781-5. doi: 10.1073/pnas.84.14.4781.

DOI:10.1073/pnas.84.14.4781
PMID:3037524
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC305189/
Abstract

RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). The replication was also completely dependent on the host DnaA function. In filter binding assays in high salt (0.5 M KCl) conditions, RepA specifically binds to both supercoiled and linear plasmid DNA containing the oriR sequence, whereas it binds to nonspecific DNA in low salt. DNase I-protection studies on a linearized DNA fragment revealed that DnaA protein specifically binds to a 9-base-pair DnaA-recognition sequence ("DnaA box") within oriR only when RepA is bound to the sequence immediately downstream of the DnaA box. These results indicate that initiation of R1 plasmid replication is triggered by interaction of RepA and DnaA proteins with the oriR sequence.

摘要

RepA是R1质粒复制的起始蛋白，它是从过量表达该蛋白的大肠杆菌菌株中纯化得到的。纯化后的RepA蛋白能在体外特异性地起始携带R1质粒复制起点（oriR）的质粒DNA的复制。该复制严格依赖于添加的RepA蛋白，不依赖于宿主RNA聚合酶，但需要其他宿主复制功能（DnaB和DnaC蛋白、单链DNA结合蛋白SSB以及DNA回旋酶）。该复制也完全依赖于宿主的DnaA功能。在高盐（0.5 M KCl）条件下的滤膜结合试验中，RepA能特异性地结合含有oriR序列的超螺旋和线性质粒DNA，而在低盐条件下它能结合非特异性DNA。对线性化DNA片段进行的DNase I保护研究表明，只有当RepA与DnaA框下游紧邻的序列结合时，DnaA蛋白才能特异性地结合oriR内一个9碱基对的DnaA识别序列（“DnaA框”）。这些结果表明，R1质粒复制的起始是由RepA和DnaA蛋白与oriR序列的相互作用触发的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/c4d9ca8636c3/pnas00279-0128-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/46d888f3b5d8/pnas00279-0126-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/27d6590cefbb/pnas00279-0126-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/c472bef78399/pnas00279-0126-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/4447a52d4b7b/pnas00279-0128-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/0702c5370626/pnas00279-0128-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/c4d9ca8636c3/pnas00279-0128-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/46d888f3b5d8/pnas00279-0126-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/27d6590cefbb/pnas00279-0126-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/c472bef78399/pnas00279-0126-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/4447a52d4b7b/pnas00279-0128-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/0702c5370626/pnas00279-0128-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dc6e/305189/c4d9ca8636c3/pnas00279-0128-c.jpg

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本文引用的文献

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Gene. 1981 Nov;15(2-3):257-71. doi: 10.1016/0378-1119(81)90135-9.
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Plasmid R1 DNA replication dependent on protein synthesis in cell-free extracts of E. coli.质粒R1 DNA复制依赖于大肠杆菌无细胞提取物中的蛋白质合成。
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Role of RepA and DnaA proteins in the opening of the origin of DNA replication of an IncB plasmid.RepA和DnaA蛋白在IncB质粒DNA复制起点解链中的作用。
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Sequencing end-labeled DNA with base-specific chemical cleavages.通过碱基特异性化学切割对末端标记的DNA进行测序。
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