Biochemical analysis of nucleosome targeting by Tn5 transposase
Biochemical analysis of nucleosome targeting by Tn5 transposase
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DOI：
10.1098/rsob.190116
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发表时间：
2019-08-01
期刊：
影响因子：
5.8
通讯作者：
Kurumizaka, Hitoshi
中科院分区：
文献类型：
--
作者：
Sato, Shoko;Arimura, Yasuhiro;Kurumizaka, Hitoshi
Tn5转座酶是一种将DNA片段整合到基因组DNA中的细菌酶，是真核生物检测基因组DNA无核小体区域的工具。然而，在染色质中，Tn5转座酶对DNA的靶向作用尚不清楚。在本研究中，我们重组了定位良好的601个二核小体，其中两个核小体与一个连接体DNA连接，并在体外用Tn5转座酶研究了二核小体中的DNA整合位点。我们发现Tn5转座酶优先靶向核小体内进出DNA区域附近。Tn5转座酶在没有连接DNA的情况下对二核小体进行了最小程度的切割，表明两个核小体之间的连接DNA对Tn5转座酶的活性很重要。在30碱基对的连接体DNA存在的情况下，Tn5转座酶除了靶向核小体的进出位点外，还靶向连接体DNA的中间。有趣的是，这种tn5靶向特性在二核小体底物中是保守的，其DNA序列与601序列不同。因此，本文报道的核小体模板中的Tn5靶向偏好为基于Tn5转座酶的基因组学方法（如ATAC-seq）的解释提供了重要信息。
Tn5 transposase is a bacterial enzyme that integrates a DNA fragment into genomic DNA, and is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes. However, in chromatin, the DNA targeting by Tn5 transposase has remained unclear. In the present study, we reconstituted well-positioned 601 dinucleosomes, in which two nucleosomes are connected with a linker DNA, and studied the DNA integration sites in the dinucleosomes by Tn5 transposase in vitro. We found that Tn5 transposase preferentially targets near the entry-exit DNA regions within the nucleosome. Tn5 transposase minimally cleaved the dinucleosome without a linker DNA, indicating that the linker DNA between two nucleosomes is important for the Tn5 transposase activity. In the presence of a 30 base-pair linker DNA, Tn5 transposase targets the middle of the linker DNA, in addition to the entry-exit sites of the nucleosome. Intriguingly, this Tn5-targeting characteristic is conserved in a dinucleosome substrate with a different DNA sequence from the 601 sequence. Therefore, the Tn5-targeting preference in the nucleosomal templates reported here provides important information for the interpretation of Tn5 transposase-based genomics methods, such as ATAC-seq.