In Vivo and In Vitro Assay for Monitoring the Topological Loading of Bacterial Condensins on DNA
In Vivo and In Vitro Assay for Monitoring the Topological Loading of Bacterial Condensins on DNA
复制标题
用于监测 DNA 上细菌凝聚蛋白拓扑负载的体内和体外测定
DOI：
10.1007/978-1-4939-9520-2_14
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发表时间：
2019
影响因子：
--
通讯作者：
and Hironori Niki
中科院分区：
文献类型：
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作者：
Koichi Yano;Koichiro Akiyama;and Hironori Niki
凝聚素在从细菌到高等生物的生命形式中染色体DNA的压缩和分离中起着重要作用。为了阐明这些作用背后的分子机制，关键是要确定如何以及在何处将凝聚素加载到染色体DNA。在这里，我们描述了在体内和体外检测监测拓扑加载的两种细菌凝聚素，Smc-ScpAB和MukBEF。这些测定中的关键步骤是用高浓度的盐洗涤样品，以区分细菌凝聚素与DNA的静电结合和拓扑结合。此外，细菌凝聚素和DNA复合物的分离防止了它们之间由于交联试剂而产生的任何不期望的相互作用。这些方法提供了可重复的和可靠的结果，拓扑结合的蛋白质，如细菌凝聚素的负载。
Condensins play essential roles in the compaction and segregation of chromosomal DNA in life forms ranging from bacteria to higher organisms. To elucidate the molecular mechanisms underlying these roles, it is crucial to determine how and where condensins are loaded to chromosomal DNA. Here, we describe in vivo and in vitro assays for monitoring the topological loading of two bacterial condensins, Smc-ScpAB and MukBEF. A key step in these assays is washing the samples with a high concentration of salt in order to discriminate between electrostatic and topological binding of the bacterial condensins to DNA. In addition, isolation of bacterial condensin and DNA complexes prevents any undesired interaction between them due to cross-linking reagents. These methodologies provide reproducible and reliable results for the loading of topologically bound proteins such as bacterial condensins.