Lindsley J E, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10485-9. doi: 10.1073/pnas.88.23.10485.
A cloned yeast TOP2 gene was modified to produce yeast DNA topoisomerase II (EC 5.99.1.3) epitopically labeled at its amino or carboxyl terminus. Limited digestion with SV8 endoprotease shows three distinct protease-sensitive sites in each polypeptide of the dimeric enzyme. These sites were mapped by immunostaining of the end-labeled proteolytic fragments resolved by SDS/polyacrylamide gel electrophoresis; two of the mapped locations were confirmed by sequencing the amino ends of two unlabeled peptic fragments. Proteolytic cleavage by SV8 endoprotease at a pair of sites corresponding to the carboxyl sides of Glu-411 and Glu-680 is modulated by the binding of the nonhydrolyzable ATP analogs adenosine 5'-[beta, gamma-imido]triphosphate (5'-adenylyl imidodiphosphate) and adenosine 5'-[gamma-thio]triphosphate: in their absence cleavage occurs predominantly at Glu-411; in the presence of either analog, cleavage occurs predominantly at Glu-680. These results are interpreted in terms of allosteric interdomainal movements in the type II DNA topoisomerase following the binding of ATP.
一个克隆的酵母TOP2基因经过修饰，可产生在其氨基或羧基末端带有表位标签的酵母DNA拓扑异构酶II（EC 5.99.1.3）。用SV8内切蛋白酶进行有限消化显示，在二聚体酶的每个多肽中有三个不同的蛋白酶敏感位点。通过对经SDS/聚丙烯酰胺凝胶电泳分离的末端标记蛋白水解片段进行免疫染色来确定这些位点；通过对两个未标记的胃蛋白酶片段的氨基末端进行测序，证实了其中两个定位位置。与Glu-411和Glu-680羧基侧相对应的一对位点处的SV8内切蛋白酶的蛋白水解切割受不可水解的ATP类似物腺苷5'-[β,γ-亚氨基]三磷酸（5'-腺苷酰亚氨基二磷酸）和腺苷5'-[γ-硫代]三磷酸结合的调节：在它们不存在时，切割主要发生在Glu-411处；在任何一种类似物存在时，切割主要发生在Glu-680处。这些结果根据ATP结合后II型DNA拓扑异构酶中的变构域间运动来解释。
