Insight into the catalytic mechanism of DNA polymerase β:: Structures of intermediate complexes
Insight into the catalytic mechanism of DNA polymerase β:: Structures of intermediate complexes
复制标题
DOI：
10.1021/bi002176j
复制
发表时间：
2001-05-08
期刊：
影响因子：
2.9
通讯作者：
Chan, MK
中科院分区：
文献类型：
--
作者：
Arndt, JW;Gong, WM;Chan, MK
已知由DNA聚合酶介导的催化反应需要两个Mg（II）离子，一个与dNTP结合相关，另一个参与化学步骤的金属离子催化。在此，我们报道了仅结合一个金属离子的DNA聚合酶的功能性中间体结构，DNA聚合酶β-DNA模板-引物-铬（LII）.2 '-脱氧胸苷5'-β，γ-亚甲基三磷酸[Cr（III）。dTMPPCP]复合物，分辨率为2.6埃。该复合物与其它聚合酶-DNA-ddNTP复合物的结构不同之处在于引物的3 ′-末端具有游离羟基。因此，这种结构代表了一种完全功能性的中间状态。通过观察结晶蛋白的生化测定中的周转以及通过测定用Mn（LT）离子浸泡Pol β晶体导致产物复合物Pol β-DNA-Cr（LII）的形成，为该论点提供了支持。五氯苯酚，其结构也已报告。这两种结构的一个重要特征是指子域是封闭的，类似于其中两个金属离子位点都被占据的其他三元络合物的结构。这些结果表明，关闭的手指亚结构域是由金属-dNTP复合物的结合之前，催化Mg 2+离子的结合特异性诱导。这使我们重新评估我们以前的证据，关于存在一个限速构象变化的Pol β的反应途径。停流研究的结果表明，没有可检测到的限速构象变化步骤。
The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in metal ion catalysis of the chemical step, Here we report a functional intermediate structure of a DNA polymerase with only one metal ion bound, the DNA polymerase beta -DNA template-primer-chromium(LII).2'-deoxythymidine 5'-beta,gamma -methylenetriphosphate [Cr(III). dTMPPCP] complex, at 2.6 Angstrom resolution. The complex is distinct from the structures of other polymerase-DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydroxyl,group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnover in biochemical assays of crystallized protein as well as from the determination that soaking Pol beta crystals with Mn(LT) ions leads to formation of the product complex, Pol beta -DNA-Cr(LII). PCP, whose structure is also reported. An important feature of both structures is that the fingers subdomain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of the fingers subdomain is induced specifically by binding of the metal-dNTP complex prior to binding of the catalytic Mg2+ ion. This has led us to reevaluate our previous evidence regarding the existence of a rate-limiting conformational change in Pol beta 's reaction pathway. The results of stopped-flow studies suggest that there is no detectable rate-limiting conformational change step.