Sequence-dependent base pair stepping dynamics in XPD helicase unwinding.
Sequence-dependent base pair stepping dynamics in XPD helicase unwinding.
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DOI：
10.7554/elife.00334
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发表时间：
2013-05-28
期刊：
影响因子：
7.7
通讯作者：
Chemla YR
中科院分区：
文献类型：
--
作者：
Qi Z;Pugh RA;Spies M;Chemla YR
解旋酶将ATP水解的化学能与核酸的定向移位和瞬时双链分离结合起来。了解解旋酶的作用机制需要了解碱基对分离的基本物理化学过程。这就需要在高时空分辨率下直接监测解旋酶活性。使用单碱基对(BP)分辨率的光钳，我们分析了参与DNA修复和转录启动的超家族2(SF2)DNA解旋酶XPD解旋酶的DNA解旋。我们发现，单体XPD以1个碱基的步长解开双链DNA，但表现出频繁的后退和经历5个碱基的后退和前进步长的构象转变。用近碱基对分辨率量化XPD步进动力学的序列依赖性，我们提供了迄今为止最有力和最直接的证据，证明解旋酶的正向单碱基对步进利用了双链的自发打开。所提出的解离机制可能是沿DNA磷酸二酯骨架移动的DNA解旋酶的普遍特征。DOI：http://dx.doi.org/10.7554/eLife.00334.001在许多细胞过程中，双螺旋必须瞬间解开，这样负责维持基因组的酶才能接触到这两条链。例如，在DNA合成过程中，DNA的两条链首先被分离，然后用作生产新链的模板。破坏、分离和解开双螺旋的作用落在被称为DNA解旋酶的酶上。在无数其他细胞过程中，解旋酶也参与分离核酸链，包括DNA修复、转录和翻译。虽然解旋酶的功能很清楚，但它们解开DNA的确切机制还不清楚。在这里，齐等人。研究了一种名为XPD的解旋酶的机制，它参与DNA修复和DNA转录为RNA的启动。他们使用光学镊子--其中激光被用来对单个DNA分子施加极小的力--在解开碱基对分辨率的DNA时跟踪XPD单个分子的活动。qi等人观察到，解旋酶一次解开一个碱基对的DNA链，但它有时后退一个碱基对，另一些时候使5个碱基对前后移动。这些倒退的频率取决于ATP的可用性和DNA的序列。由于数据的高分辨率，齐等人。能够将这些步进动力学与碱基对水平的DNA序列相关联。虽然一些解旋酶活跃地分离链，利用来自ATP的能量来打破碱基对之间的氢键，齐等人说。显示XPD似乎利用了碱基对之间自发产生的瞬间分离。齐等人的工作不仅为XPD在DNA修复和转录中的作用提供了见解。提出了一种可用于探索其他解旋酶机制的方法。鉴于这里描述的解离机制可能是与XPD相关的酶的普遍特征，目前的工作可能有助于揭示涉及XPD样解旋酶的其他一些细胞过程，如同源DNA重组、链间交联修复和准确的染色体分离。DOI：http://dx.doi.org/10.7554/eLife.00334.002
Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones. DOI: http://dx.doi.org/10.7554/eLife.00334.001 During many cellular processes, the double helix must be transiently unwound so that the enzymes responsible for maintaining the genome can access the two strands. During DNA synthesis, for instance, the two strands of DNA are first separated and then used as templates for the production of new strands. The role of destabilizing, separating and unwinding the double helix falls to enzymes known as DNA helicases. Helicases are also involved in separating strands of nucleic acids during myriad other cellular processes, including DNA repair, transcription and translation. While the functions of helicases are clear, the precise mechanisms by which they unwind DNA are not. Here, Qi et al. have investigated the mechanism of a helicase called XPD, which is involved in DNA repair and the initiation of transcription of DNA into RNA. Using optical tweezers—in which a laser beam is used to exert extremely small forces on a single DNA molecule—they followed the activity of individual molecules of XPD as they unwound DNA with base pair resolution. Qi et al. observed that the helicase unwinds DNA strands 1 base pair at a time, but that it sometimes moves backwards by 1 base pair and at other times makes 5 base pair backward and forward steps. The frequency of these backwards steps depends on the availability of ATP, and the sequence of the DNA. Due to the high resolution of the data, Qi et al. were able to correlate these stepping dynamics with the DNA sequence with base pair level accuracy. While some helicases actively separate the strands, using energy derived from ATP to break the hydrogen bonds between pairs of bases, Qi et al. showed that XPD appears to take advantage of momentary separations that arise spontaneously between base pairs. As well as providing insights into the role of XPD in DNA repair and transcription, the work of Qi et al. presents a method that could be used to explore the mechanisms of other helicases. Given that the unwinding mechanism described here is likely to be a universal feature of enzymes related to XPD, the current work could shed light on a number of other cellular processes involving XPD-like helicases, such as homologous DNA recombination, inter-strand cross-link repair, and accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.00334.002