DNA binding activity of the proximal C-terminal domain of rat DNA topoisomerase IIβ is involved in ICRF-193-induced closed-clamp formation.
DNA binding activity of the proximal C-terminal domain of rat DNA topoisomerase IIβ is involved in ICRF-193-induced closed-clamp formation.
复制标题
大鼠DNA拓扑异构酶IIβ的近端C末端结构域的DNA结合活性参与ICRF-193诱导的闭合夹的形成。
DOI：
10.1371/journal.pone.0239466
复制
发表时间：
2020
期刊：
影响因子：
3.7
通讯作者：
Ikeda S
中科院分区：
文献类型：
--
作者：
Kawano S;Fujimoto K;Yasuda K;Ikeda S
DNA拓扑异构酶II（topoisomerase II，topo II）是一种通过DNA切割和重新连接来调节DNA拓扑结构的必需酶。在脊椎动物中，有两种同工酶，α和β。同工酶的C-末端结构域（CTD）在α和β之间显示出低程度的序列同源性，其参与每种同工酶特异性的细胞内行为。Topo IIβ的CTD被认为参与Topo II调节。Topo IIβ通过RNA与CTD中称为C-末端调节结构域（CRD）的50个残基区域结合，在核仁中保持非活性状态。尽管体外生化分析表明，拓扑异构酶IIβ的CTD具有DNA结合活性，但CTD是否影响核质中的催化反应尚不清楚。在这里，我们表明，近端CTD（以下简称为pCTD）的大鼠拓扑IIβ，包括CRD，参与了核质中的催化反应。我们通过体外连接试验和电泳迁移率变动试验鉴定了pCTD为具有DNA结合活性的结构域。荧光漂白后恢复（FRAP）分析表明，缺失突变体（ΔpCTD）在核质中的迁移率高于野生型，表明pCTD也影响了topo IIβ的核动力学。ICRF-193是拓扑异构酶II的催化抑制剂之一，可诱导拓扑异构酶II的闭合钳中间体的形成。用ICRF-193处理ΔpCTD显著降低了闭合钳形成的效率。总之，我们的数据表明，topo IIβ通过pCTD与DNA的结合是核质中催化反应所必需的。
DNA topoisomerase II (topo II) is an essential enzyme that regulates DNA topology by DNA cleavage and re-ligation. In vertebrates, there are two isozymes, α and β. The C-terminal domain (CTD) of the isozymes, which shows a low degree of sequence homology between α and β, is involved in each isozyme-specific intracellular behavior. The CTD of topo IIβ is supposedly involved in topo II regulation. Topo IIβ is maintained in an inactive state in the nucleoli by the binding of RNA to the 50-residue region termed C-terminal regulatory domain (CRD) present in the CTD. Although in vitro biochemical analysis indicates that the CTD of topo IIβ has DNA binding activity, it is unclear whether CTD influences catalytic reaction in the nucleoplasm. Here, we show that the proximal CTD (hereafter referred to as pCTD) of rat topo IIβ, including the CRD, is involved in the catalytic reaction in the nucleoplasm. We identified the pCTD as a domain with DNA binding activity by in vitro catenation assay and electrophoretic mobility shift assay. Fluorescence recovery after photo-bleaching (FRAP) analysis of pCTD-lacking mutant (ΔpCTD) showed higher mobility in nucleoplasm than that of the wild-type enzyme, indicating that the pCTD also affected the nuclear dynamics of topo IIβ. ICRF-193, one of the topo II catalytic inhibitors, induces the formation of closed-clamp intermediates of topo II. Treatment of ΔpCTD with ICRF-193 significantly decreased the efficiency of closed-clamp formation. Altogether, our data indicate that the binding of topo IIβ to DNA through the pCTD is required for the catalytic reaction in the nucleoplasm.
登录
查看更多内容
影响因子：
16.6
作者：
Chen SF;Huang NL;Lin JH;Wu CC;Wang YR;Yu YJ;Gilson MK;Chan NL
通讯作者：
Chan NL
影响因子：
4.6
作者：
Morotomi-Yano K;Saito S;Adachi N;Yano KI
通讯作者：
Yano KI
拓扑异构酶iialpha和IIBETA的C末端区域确定体内酶的同工型特异性功能。
影响因子：
14.9
作者：
Linka, Rene M;Porter, Andrew C G;Volkov, Arsen;Mielke, Christian;Boege, Fritz;Christensen, Morten O
通讯作者：
Christensen, Morten O
影响因子：
64.8
作者：
Dong, Ken C.;Berger, James M.
通讯作者：
Berger, James M.
影响因子：
4.8
作者：
Morris, SK;Baird, CL;Lindsley, JE
通讯作者：
Lindsley, JE