Structure-function analysis of a molecular switch for long-range diffusion on DNA

描述(申请人提供)：解旋酶由一大类酶组成，从“经典”到“假解旋酶”，它们在基因组维持中起着重要作用。假解旋酶在从癌症、染色质重塑到远距离DNA位点之间的远程通讯等生物学过程中一直是研究的对象。虽然一些假解旋酶是骨骼马达或转位酶，它们消耗数百个ATP分子在DNA/RNA上进行连续运动，但另一些却被证明是只水解几个ATP来转换结构状态的“分子开关” 远程扩散。这些分子开关在从核苷酸切除修复到错配修复的一系列过程中都很重要，但它们的作用机制仍然是个谜。III型限制性内切酶提供了研究假解旋酶活性的理想系统，因为所有的酶功能都集成在同一个全酶复合体中，并且不需要额外的蛋白质辅助因子。我们在这里提出了一系列实验，将X射线结晶学与最先进的单分子和系综测量相结合，以解释类型III RE家族的原型EcoP15I如何从一种状态转换到另一种状态，从而在DNA上实现长寿命滑动。在目标1中，我们将获得EcoP15I的第一个三维结构信息。除了天然的Eco 15I/DNA复合体外，我们还将确定在ADP和ATP类似物存在下的结构，以及突触或“碰撞”复合体中的酶的结构。拟议的结构研究是关于III型限制性内切酶的第一次，也是关于与双链DNA结合的解旋酶的第二次。在目标2中，我们将得出一个动力学框架 构造结果的解释。在该结构的指导下，我们将对EcoP15I与DNA和ATP相互作用过程中的动力学进行单分子和系综荧光共振能量转移测量。我们将利用一种专门建造的磁镊子-全内反射荧光(MT-TIRF)显微镜，它可以可视化单个荧光标记的蛋白质沿着在磁场中拉伸的DNA滑动。这将得到使用停流的毫秒时间分辨率荧光分析以及新的基于Biacore的DNA解离分析的补充。总之，拟议的“实时”分析将补充结构研究，并为DNA代谢中依赖于ATP的分子开关的反应路径提供前所未有的新细节。

DESCRIPTION (provided by applicant): Helicases comprise a large group of enzymes, from "classical" to "pseudo-helicases", which play important roles in genome maintenance. The pseudo-helicases have been the subject intense investigation in biological processes ranging from cancer, chromatin remodeling, to long-range communication between distant DNA sites. While some of pseudo-helicases are bone-fide motors or translocases that consume hundreds of ATP molecules to processively move on the DNA/RNA, others are turning out to be "molecular switches" that hydrolyze just a few ATPs to switch structural states for long-range diffusion. These molecular switches are important in processes ranging from nucleotide excision repair to mismatch repair, but their mechanism of action remains mysterious. The Type III restriction enzymes (REs) offer the ideal system to investigat pseudo-helicase activity because all of the enzymatic functions are integrated in the same holoenzyme complex and no additional protein cofactors are required. We propose here a set of experiments combining X-ray crystallography with state-of-the-art single-molecule and ensemble measurements to elucidate how, EcoP15I, a prototype of the Type III RE family, transitions from one state to another for long-lived sliding on DNA. In Aim 1, we wil derive the first 3-D structural information on EcoP15I. In addition to the native Ecop15I/DNA complex, we will determine structures in the presence of ADP and ATP analogues, as well as structure of the enzyme in synaptic or "collision" complex. The proposed structural studies are the first for a Type III restriction enzyme, and only the second for a helicase bound to double-stranded DNA. In aim 2, we will derive a kinetic framework for the interpretation of structural results. Guided by the structure, we will perform single molecule and ensemble fluorescence resonance energy transfer measurements of EcoP15I dynamics during interaction with DNA and ATP. We will take advantage of a specially built magnetic tweezers-total internal reflection fluorescence (MT-TIRF) microscope that can visualize single fluorescently- labeled proteins sliding along DNA stretched within a magnetic field. This will be complemented by millisecond time resolution fluorescent assays using stopped flow, as well as new Biacore-based DNA dissociation assays. Together, the proposed "real-time" assays will complement the structural studies and provide unprecedented new details on the reaction pathway of an ATP-dependent molecular switch in DNA metabolism.