Purification and characterization of human DNA topoisomerase IIIα
Purification and characterization of human DNA topoisomerase IIIα
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DOI：
10.1093/nar/27.12.2443
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发表时间：
1999-06-15
影响因子：
14.9
通讯作者：
Riou, JF
中科院分区：
文献类型：
--
作者：
Goulaouic, H;Roulon, T;Riou, JF
人拓扑异构酶Ⅲ α（hTopo Ⅲ alpha）是最近发现的人类拓扑异构酶IA亚家族的第一个成员，其中心结构域与酵母拓扑异构酶Ⅲ高度同源，但总体结构更接近大肠杆菌DNA拓扑异构酶Ⅰ。为了确定hTopo III α的性质，与其他拓扑异构酶IA亚家族成员的性质相比，我们将该酶与活性位点突变体Y337 F一起纯化至接近同质。我们表明hTopo III α能够以分布的方式松弛负超螺旋DNA，导致初始底物的完全消失和中间拓扑异构体的出现，这种DNA松弛活性是镁依赖性的，尽管低浓度的MgCl 2足以获得有效的催化。P-32-转移实验证明，hTopo III α能够切割单链寡核苷酸并共价结合至切割的DNA的5 ′-末端。加入0.5 M NaCl逆转反应，导致寡核苷酸的再连接，利用几种不同的单链寡核苷酸的实验使我们能够绘制几个切割位点，并推导出DNA切割的共有序列（CANNN向下箭头），这与Topo IA亚家族的其他成员不同。
Human topoisomerase III alpha (hTopo III alpha), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo III alpha, compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F, We show that hTopo III alpha is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers, This DNA relaxation activity is magnesium-dependent, although a low concentration of MgCl2 is sufficient to obtain efficient catalysis. P-32-transfer experiments demonstrated that hTopo III alpha is able to cleave a single-stranded oligonucleotide and to bind covalently to the 5'-end of the cleaved DNA, Addition of 0.5 M NaCl reverses the reaction, leading to the religation of the oligonucleotide, Experiments utilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleavage (CANNN down arrow), which is different from that for other members of the Topo IA subfamily.