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在含有滚环复制质粒的指数生长细菌中以核苷酸分辨率测定特定DNA链的不连续性。

Determination of specific DNA strand discontinuities with nucleotide resolution in exponentionally growing bacteria harboring rolling circle-replicating plasmids.

作者信息

Grohmann E, Zechner E L, Espinosa M

机构信息

Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.

出版信息

FEMS Microbiol Lett. 1997 Jul 15;152(2):363-9. doi: 10.1111/j.1574-6968.1997.tb10453.x.

Abstract

Plasmid replication by the rolling circle mechanism and conjugative transfer of plasmids require the generation of a specific strand discontinuity in the DNA. In both processes cleavage at the so-called nic site is catalyzed by plasmid-encoded proteins. The strand discontinuities at the conjugative origins of transfer of plasmid pE194 and pMV158 were determined in Bacillus subtilis and Streptococcus pneumoniae, respectively, with a recently developed runoff DNA synthesis assay. The positions of intracellular cleavage within the respective transfer origins were shown to coincide with the site predicted for pE194 and with the nic site determined in vitro for pMV158. For pMV158, the influence of a mutation in the S. pneumoniae polA gene on the efficiency of replication was investigated. In addition, the nic site within the double-stranded origin of the-rolling circle-replicating plasmid pMV158 in S. pneumoniae as well as that of pFX2 in Escherichia coli was mapped with nucleotide resolution.

摘要

通过滚环机制进行的质粒复制以及质粒的接合转移需要在DNA中产生特定的链断裂。在这两个过程中,所谓切口位点(nic位点)的切割是由质粒编码的蛋白质催化的。分别在枯草芽孢杆菌和肺炎链球菌中,利用最近开发的径流DNA合成测定法确定了质粒pE194和pMV158接合转移起始位点处的链断裂情况。结果表明,各自转移起始位点内细胞内切割的位置与pE194预测的位点以及体外确定的pMV158的nic位点一致。对于pMV158,研究了肺炎链球菌polA基因突变对复制效率的影响。此外,以核苷酸分辨率绘制了肺炎链球菌中滚环复制质粒pMV158以及大肠杆菌中pFX2双链起始位点内的nic位点。

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