Global conformational dynamics of a Y-family DNA polymerase during catalysis.
Global conformational dynamics of a Y-family DNA polymerase during catalysis.
复制标题
DOI：
10.1371/journal.pbio.1000225
复制
发表时间：
2009-10
期刊：
影响因子：
9.8
通讯作者：
Suo Z
中科院分区：
文献类型：
--
作者：
Xu C;Maxwell BA;Brown JA;Zhang L;Suo Z
对蛋白质和DNA聚合过程中DNA构象变化的高分辨率分析，建立了DNA聚合酶各个结构域的酶功能和构象动力学之间的关系。复制的DNA聚合酶因DNA损伤而停滞，而新发现的Y家族DNA聚合酶被招募来拯救这些停滞的复制分叉，从而提高细胞的存活。Y-家族DNA聚合酶具有低保真性和可加工性的特点，能够绕过不同类别的DNA损伤。各种动力学和结构研究已经建立了所有DNA聚合酶共有的最小反应途径，尽管构象中间体还没有很好地定义。此外，任何DNA聚合酶催化的核苷酸掺入的限速步骤的识别一直是一个长期争论的问题。通过监测催化过程中每个结构域和DNA中多个位点的时间相关荧光共振能量转移(FRET)信号的变化，我们展示了一个模型Y家族酶的全球构象变化的实时图像：来自Sulfolobus solfararicus的DNA聚合酶IV(DPO4)。我们的结果为假想的DNA易位事件提供了证据，随后是催化前蛋白质构象的快速变化，以及随后缓慢的、化学后的蛋白质构象变化。令人惊讶的是，DNA易位步骤是由正确的核苷酸结合引起的。此外，我们还确定了蛋白质构象转变的方向、速率和激活能垒，表明DPO4的四个结构域是以同步方式移动的。这些结果最终表明，与结构域移动相关的化学前构象变化太快，不能作为速率限制步骤。相反，活性中心残基的重排限制了正确的核苷酸掺入速度。总而言之，DPO4的构象动力学提供了关于结构域间运动如何与酶功能相关的见解，以及它们与复制分叉上的其他蛋白质的协同作用。DNA聚合酶对基因组DNA的忠实复制对于维持生物体的遗传完整性至关重要。如果DNA受损，专门的病变旁路DNA聚合酶被招募来纠正DNA中的错误。各种动力学和结构研究已经建立了所有DNA聚合酶共有的最小动力学机制。这种机制包括几个步骤，涉及离散的蛋白质构象变化。然而，构象动力学和酶功能之间的相互关系仍然不清楚，在核苷酸掺入过程中的限速步骤的识别也一直存在争议。在这项研究中，我们监测了在正确的核苷酸掺入过程中病变旁路聚合酶结构域的运动方向和速率。我们的研究提供了几个重要的发现。首先，正确的核苷酸结合会引发一个快速且令人惊讶的DNA易位事件。其次，聚合酶的所有四个结构域在聚合反应前后都以同步的方式快速移动。第三，活性位点残基的重新定位是正确的核苷酸掺入过程中的限速步骤。因此，聚合酶和聚合酶结合的DNA底物的运动与催化紧密相连。
High-resolution analysis of protein, and DNA conformational changes during DNA polymerization, established relationships between the enzymatic function and conformational dynamics of individual domains for a DNA polymerase. Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork. Faithful replication of genomic DNA by DNA polymerases is crucial for maintaining the genetic integrity of an organism. If DNA becomes damaged, specialized lesion-bypass DNA polymerases are recruited to correct errors in the DNA. A variety of kinetic and structural studies have established a minimal kinetic mechanism common to all DNA polymerases. This mechanism includes several steps involving discrete protein conformational changes. However, the inter-relationship between conformational dynamics and enzymatic function has remained unclear, and identification of the rate-limiting step during nucleotide incorporation has been controversial. In this study, we monitored the directions and rates of motion of domains of a lesion-bypass polymerase during correct nucleotide incorporation. Our study provides several significant findings. First, the binding of a correct nucleotide induces a fast and surprising DNA translocation event. Second, all four domains of the polymerase rapidly move in a synchronized manner before and after the polymerization reaction. Third, repositioning of active site residues is the rate-limiting step during correct nucleotide incorporation. Thus, the motions of the polymerase and the polymerase-bound DNA substrate are tightly coupled to catalysis.
登录
查看更多内容
影响因子：
64.5
作者：
Franklin, MC;Wang, JM;Steitz, TA
通讯作者：
Steitz, TA
DOI：
10.1073/pnas.0503388102
发表时间：
2005-12-06
影响因子：
11.1
作者：
Bu, ZM;Biehl, R;Callaway, DJE
通讯作者：
Callaway, DJE
影响因子：
16.8
作者：
Garcia-Diaz, M;Bebenek, K;Pedersen, LC
通讯作者：
Pedersen, LC
影响因子：
2.9
作者：
Joyce, Catherine M.;Potapova, Olga;Grindley, Nigel D. F.
通讯作者：
Grindley, Nigel D. F.
影响因子：
2.9
作者：
Fiala, KA;Suo, Z
通讯作者：
Suo, Z