Lue N, Sharma A, Mondragón A, Wang J C
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Structure. 1995 Dec 15;3(12):1315-22. doi: 10.1016/s0969-2126(01)00269-6.
Type I DNA topoisomerases, divided mechanistically into two subfamilies, are ubiquitous enzymes that participate in replication and transcription. In addition to its role in these fundamental processes, the biological importance of eukaryotic DNA topoisomerase I is underscored by its identification as the target of the antitumor alkaloid camptothecin. An understanding of the mechanism of catalysis and interactions with camptothecin and other drugs has been hampered by a lack of detailed structural information.
The three-dimensional structure of a 26 kDA fragment (residues 135 to about 363) of Saccharomyces cerevisiae DNA topoisomerase I has been determined at 1.9 A resolution. The fragment has a novel architecture comprising a concave platform and a pair of outlying V-shaped helices. Photocrosslinking and protein footprinting experiments show that the positively charged concave surface and the junction region of the V-shaped pair of helices contact DNA in the enzyme-DNA complex.
Crystallographic, biochemical and genetic data indicate that this 26 kDa fragment of yeast DNA topoisomerase I is involved in complex formation between the enzyme and DNA, and probably also in camptothecin-enzyme-DNA ternary complex formation. A molecular model for protein-DNA interaction based on these data is proposed. The bipartite DNA-binding regions of the 26 kDa fragment may enable eukaryotic DNA topoisomerase I to adapt to sequence-dependent structural variations in its DNA substrates.
I型DNA拓扑异构酶在机制上分为两个亚家族，是参与复制和转录的普遍存在的酶。除了在这些基本过程中的作用外，真核生物DNA拓扑异构酶I作为抗肿瘤生物碱喜树碱的靶点，其生物学重要性也得到了凸显。由于缺乏详细的结构信息，对其催化机制以及与喜树碱和其他药物相互作用的理解受到了阻碍。
酿酒酵母DNA拓扑异构酶I的一个26 kDa片段（残基135至约363）的三维结构已在1.9 Å分辨率下确定。该片段具有一种新颖的结构，包括一个凹面平台和一对外围的V形螺旋。光交联和蛋白质足迹实验表明，带正电荷的凹面以及V形螺旋对的连接区域在酶 - DNA复合物中与DNA接触。
晶体学、生化和遗传学数据表明，酵母DNA拓扑异构酶I的这个26 kDa片段参与了酶与DNA之间的复合物形成，可能还参与了喜树碱 - 酶 - DNA三元复合物的形成。基于这些数据提出了一个蛋白质 - DNA相互作用的分子模型。26 kDa片段的二分DNA结合区域可能使真核生物DNA拓扑异构酶I能够适应其DNA底物中依赖序列的结构变化。
