Reconstitution of enzymatic activity by the association of the cap and catalytic domains of human topoisomerase I
Reconstitution of enzymatic activity by the association of the cap and catalytic domains of human topoisomerase I
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DOI：
10.1074/jbc.m205302200
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发表时间：
2002-08-23
影响因子：
4.8
通讯作者：
Champoux, JJ
中科院分区：
文献类型：
--
作者：
Yang, Z;Champoux, JJ
当人类拓扑异构酶I结合DNA时，酶的两个相反的裂片，帽区（氨基酸，残基175-433）和催化结构域（Deltacap，残基433至COOH末端）紧紧地夹在DNA螺旋周围，形成预切割复合物。尽管Deltacap包含所有已知的催化重要残基，并且与完整的酶具有相似的亲和力结合DNA，但该片段缺乏催化活性。然而，Deltacap和topo31的混合物（残基175-433）通过质粒DNA弛豫和自杀式切割测定重建酶活性。尽管topo31和Deltacap之间形成的活性复合物太不稳定，即使在DNA存在的情况下也无法通过下拉实验检测到，但topo31与Deltacap的关联仍然存在，并且在复合物通过自杀式切割催化DNA与Deltacap的共价附着后可以检测到。自杀式切割后从Deltacap-DNA中去除topo3表明，与切割反应不同，宗教不需要蛋白质的帽区。这些结果表明，酶的催化区域的激活裂解需要DNA结合和蛋白质的帽区存在。
When human topoisomerase I binds DNA, two opposing lobes in the enzyme, the cap region (amino acid, residues 175-433) and the catalytic domain (Deltacap, residues 433 to the COOH terminus) clamp tightly around the DNA helix to form the precleavage complex. Although Deltacap contains all of the residues known to be important for catalysis and binds DNA with an affinity similar to that of the intact enzyme, this fragment lacks catalytic activity. However, a mixture of Deltacap and topo31 (residues 175-433) reconstitutes enzymatic activity as measured by plasmid DNA relaxation and suicide cleavage assays. Although the formation of an active complex between topo31 and Deltacap is too unstable to be detected by pull-down experiments even in the presence of DNA, the association of topo31 with Deltacap persists and is detectable after the complex catalyzes the covalent attachment of the DNA to Deltacap by suicide cleavage. Removal of topo3 from Deltacap-DNA after suicide cleavage reveals that, unlike the cleavage reaction, religation does not require the cap region of the protein. These results suggest that activation of the catalytic domain of the enzyme for cleavage requires both DNA binding and the presence of the cap region of the protein.