Kinetic analysis of DNA compaction by mycobacterial integration host factor at the single-molecule level
Kinetic analysis of DNA compaction by mycobacterial integration host factor at the single-molecule level
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DOI：
10.1016/j.tube.2019.101862
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发表时间：
2019-12-01
期刊：
影响因子：
3.2
通讯作者：
Fu, Yu Vincent
中科院分区：
文献类型：
--
作者：
Chen, Yuanyuan;Zhan, Zhengyan;Fu, Yu Vincent
核相关蛋白（Nucleoid-associated proteins，NAPs）在染色体的凝聚和组织中起重要作用。分枝杆菌整合宿主因子（mIHF）是迄今为止发现的少数分枝杆菌NAP之一。通过原子力显微镜观察，mIHF具有刺激分枝杆菌噬菌体L5整合和将DNA压缩成类核或更高级丝状结构的能力。在本研究中，M.具有mIHF功能所必需的序列的丝霉亲和素IHF（MsIHF）以不依赖于序列的方式结合30-bp dsDNA片段，并在生物层干涉（BLI）实验中显示对离子强度的敏感性。利用全内反射荧光显微镜（TIRFM）在单分子水平上观察MsIHF的DNA压缩过程。MsIHF在0.25和1.0 μ M的浓度下有效地将λ DNA压缩成高度浓缩的结构，并且包装比高于10。进一步的动力学分析显示MsIHF以三步机制压缩DNA，该机制由两个压缩步骤组成，其中两个压缩步骤具有由滞后步骤分开的不同压缩速率。本研究将有助于我们更好地了解分枝杆菌染色体DNA组织的机制。
Nucleoid-associated proteins (NAPs) play an important role on chromosome condensation and organization. Mycobacterial integration host factor (mIHF) is one of the few mycobacterial NAPs identified so far. mIHF has the ability to stimulate mycobacteriophage L5 integration and compact DNA into nucleoid-like or higher order filamentous structures by atomic force microscopy observation. In this study, M. smegmatis IHF (MsIHF), which possesses the sequence essential for mIHF's functions, binds 30-bp dsDNA fragments in a sequence-independent manner and displays sensitivity to ion strength in bio-layer interferometry (BLI) experiments. The DNA compaction process of MsIHF was observed at the single-molecule level using the total internal reflection fluorescence microscopy (TIRFM). MsIHF efficiently compacted lambda DNA into a highly condensed structure with the concentration of 0.25 and 1.0 mu M, and the packing ratios were higher than 10. Further kinetic analysis revealed MsIHF compacts DNA in a three-step mechanism, which consists of two compaction steps with different compacting rates separated by a lag step. This study would help us better understand the mechanisms of chromosomal DNA organization in mycobacteria.