Goulaouic H, Roulon T, Flamand O, Grondard L, Lavelle F, Riou J F
Rhône-Poulenc Rorer, Centre de recherche de Vitry-Alfortville, 13 quai Jules Guesde, BP14, 94403 Vitry sur Seine Cedex, France.
Nucleic Acids Res. 1999 Jun 15;27(12):2443-50. doi: 10.1093/nar/27.12.2443.
Human topoisomerase IIIalpha (hTopo IIIalpha), 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 IIIalpha, 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 IIIalpha 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 MgCl2is sufficient to obtain efficient catalysis. 32P-transfer experiments demonstrated that hTopo IIIalpha 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 oligo-nucleotide. Experiments utilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleavage (CANNN downward arrow), which is different from that for other members of the Topo IA subfamily.
人拓扑异构酶IIIα（hTopo IIIα）是最近在人类中鉴定出的拓扑异构酶IA亚家族的首个成员，其中心结构域与酵母拓扑异构酶III高度同源，但整体结构更接近大肠杆菌DNA拓扑异构酶I。为了确定hTopo IIIα与其他拓扑异构酶IA亚家族成员相比的特性，我们将该酶与活性位点突变体Y337F一起纯化至接近均一状态。我们发现hTopo IIIα能够以分布方式松弛负超螺旋DNA，导致初始底物完全消失并出现中间拓扑异构体。这种DNA松弛活性依赖于镁，尽管低浓度的MgCl2就足以实现高效催化。32P转移实验表明，hTopo IIIα能够切割单链寡核苷酸并与切割后DNA的5'端共价结合。加入0.5 M NaCl可使反应逆转，导致寡核苷酸重新连接。利用几种不同的单链寡核苷酸进行的实验使我们能够绘制出几个切割位点，并推断出DNA切割的共有序列（CANNN向下箭头），这与拓扑异构酶IA亚家族的其他成员不同。
