Target DNA search by zinc-finger proteins

描述(申请人提供)：在真核细胞的细胞核中，转录因子和DNA修复/修饰酶位于数十亿个DNA碱基对之间的特定序列或结构特征，以及许多其他与DNA结合的蛋白质的存在。该项目研究团队的长期目标是了解是什么决定了蛋白质在DNA上定位其靶点的过程的效率，以便将这一知识应用于人类疾病和紊乱的治疗方法的开发。本项目的总体目标是在分子和原子水平上阐明锌指蛋白的DNA扫描机制。基于亲和力的Cys2-His2型锌指具有理想的DNA结合特异性，已广泛应用于人工基因控制/操作。事实上，基于锌指核酸酶技术的人类基因治疗正处于1期和2期临床试验阶段。然而，越来越多的报告表明，尽管人工锌指蛋白与标记物DNA位点有很高的亲和力，但仍存在动力学缺陷。这是人造锌指蛋白成功应用于治疗的瓶颈。研究小组的初步研究表明，这个问题可以通过基于DNA扫描机制知识的蛋白质工程来解决。最近，研究小组发现，当蛋白分子扫描DNA时，Egr-1锌指蛋白经历了两种不同的构象状态，称为搜索和识别模式。在目前的项目中，研究小组将进行研究，以检验中心假设，即搜索和识别模式之间的平衡是锌指蛋白搜索靶DNA的动力学效率的主要决定因素。这个项目将追求以下三个具体目标：1)描绘锌指蛋白如何扫描DNA；2)了解锌指蛋白如何绕过DNA上的障碍；3)了解锌指蛋白如何取代靶点上的其他蛋白质。对于这些特定的目标，研究小组将使用生物物理和生化方法以及诱变来改变搜索和识别模式之间的平衡。核磁共振波谱将被用来在原子水平上研究DNA扫描的动力学。荧光和生化方法将被用来在分子水平上表征锌指蛋白在目标DNA搜索过程中的动力学和热力学性质。目前的项目将大大加深我们对蛋白质DNA扫描的理解。该项目还将改善锌指蛋白的动力学性质，从而促进其在人类治疗和其他生物医学应用中的应用。

DESCRIPTION (provided by applicant): In the nuclei of eukaryotic cells, transcription factors and DNA repair/modifying enzymes locate particular sequences or structural signatures among billions of DNA base pairs and in the presence of many other proteins bound to DNA. The long-term goal for the research team of this project is to understand what dictates the efficiency in th processes whereby the proteins locate their target sites on DNA in order to apply this knowledge to the development of therapeutics for human diseases and disorders. The overall objective in the current project is to elucidate DNA-scanning mechanisms for zinc-finger proteins at both the molecular and atomic levels. Affinity-based engineering of Cys2-His2-type zinc fingers that exhibit desired DNA-binding specificity has gained popularity for artificial gene control/manipulation. In fact, human gene therapy based on the zinc-finger nuclease technology is in phase 1 and 2 clinical trials. However, there are an increasing number of reports that suggest kinetic defects in artificial zinc-finger proteins despite their high affinities for the taget DNA sites. This represents a bottleneck for successful therapeutic applications of artificial zinc-finger proteins. Preliminary studies by the research team suggest that this problem could be resolved via protein engineering based on knowledge of the DNA-scanning mechanisms. Recently the research team found that the Egr-1 zinc-finger protein undergoes two conformationally distinct states termed the search and recognition modes while the protein molecule scans DNA. In the current project, the research team will conduct research to test the central hypothesis that the balance between the search and recognition modes is a major determinant of the kinetic efficiency in target DNA search by zinc-finger proteins. The following three specific aims will be pursued in this project: 1) to delineate how zinc-finger proteins scan DNA; 2) to understand how zinc-finger proteins bypass obstacles on DNA; and 3) to understand how zinc-finger proteins displace other proteins from the target sites. For these specific aims, the research team will use biophysical and biochemical approaches along with mutagenesis to shift the equilibrium between the search and recognition modes. NMR spectroscopy will be used to investigate the dynamics of DNA scanning at an atomic level. Fluorescence and biochemical methods will be used to characterize the kinetic and thermodynamic properties of the zinc-finger proteins in the target DNA search process at a molecular level. The current project will substantially deepen our understanding of DNA scanning by proteins. This project will also enable improvement of kinetic properties of zinc-finger proteins, and thereby boost their applications to human therapeutics and other biomedical applications.