Structural Biology Studies of a Large DNA Repair Complex

我们的研究计划围绕着理解结构，动力学和 功能我们特别感兴趣的是了解这种关系，在大分子 组装体，这在过去一直是详细的结构和动力学研究的障碍。我们 策略是将高分辨率甲基基核磁共振（NMR）光谱， 它能够探测蛋白质及其复合物的大小高达~1 MDa，具有生物化学和 生物物理技术，以更好地了解功能。我们目前的重点是普遍保存和 必需DNA双链断裂（DSB）修复复合物Mre 11-Rad 50-Nbs 1（MRN）。这种蛋白质 复合物是检测DNA双链断裂并启动其修复过程的核心。几种疾病 在MRN中已经注意到突变，其引起免疫缺陷、发育和 神经退行性疾病和某些癌症的易感性。Mre 11中的其他散发性突变 和Rad 50在许多不同的癌症中被发现。细菌和古细菌模型系统， 缺乏Nbs 1，一直是现有X射线晶体学和生物化学研究的焦点 研究表明，蛋白质运动在编排Mre 11的各种功能中发挥作用， Rad 50（MR）核心复合物。然而，关于蛋白质结构的相互作用， 运动以及这些运动如何与MR活动相关并控制MR活动。在未来五年，我们的目标是确定 关键MR组件的溶液状态模型，其具有模拟不同类型的 DNA双链断裂，并表征这些复合物内发生的蛋白质动力学。基于NMR 研究将辅以各种体外生物物理和生物化学技术， 探针结构域运动和广泛的MR活动，以及在酵母体内的研究， 这些运动和活动纳入整体DNA DSB修复的背景下。我们还将扩展这些研究 包括MR中的疾病突变，这不仅可以让我们了解这些改变是如何发生的， 破坏MR功能并导致疾病，但也将为探测结构提供额外的途径， 动力学和功能关系。我们的长期目标是超越 核心MR复合体。尽管本文提出的MR研究将在简化的结构上进行， Rad 50，它已被用于所有以前的X射线晶体学研究的MR，我们的目标是执行，为 第一次，类似的研究使用全长Rad 50。总的来说，我们的研究计划旨在更好地 了解大分子组装如何使用蛋白质运动来调节它们的功能， 在将我们的程序应用于MRN的过程中，我们将确定大小尺度运动的影响， 在控制MRN介导的DNA DSB修复的第一步。

Our research program revolves around understanding the relationship between structure, dynamics, and function. We are particularly interested in understanding this relationship in large macromolecular assemblies, which have been recalcitrant to detailed structure and dynamics studies in the past. Our strategy is to couple high-resolution methyl-based nuclear magnetic resonance (NMR) spectroscopy, which is capable of probing proteins and their complexes up to ~1 MDa in size, with biochemical and biophysical techniques to better understand function. Our current focus is the universally conserved and essential DNA double strand break (DSB) repair complex Mre11-Rad50-Nbs1 (MRN). This protein complex is at the heart of detecting DNA DSBs and initiating the process of their repair. Several disease mutations have been noted in MRN, which give rise to immunodeficiency, developmental and neurodegenerative disorders, and a predisposition to certain cancers. Other sporadic mutations in Mre11 and Rad50 have been found in a number of different cancers. Bacterial and archaeal model systems, which lack Nbs1, have been the focus of the existing body of X-ray crystallography and biochemical studies that suggest a role for protein motions in choreographing the various functions of the Mre11- Rad50 (MR) core complex. Yet, many questions still remain about the interplay of protein structures and motions and how these relate to and control MR activity. Over the next five years, our goal is to determine solution state models of key MR assemblies complete with substrate DNAs that mimic different types of DNA DSBs and to characterize the protein dynamics that occur within these complexes. The NMR-based studies will be complemented with a variety of in vitro biophysical and biochemical techniques to further probe domain motions and the wide array of MR activities, as well as in vivo studies in yeast to place these motions and activities into the context of overall DNA DSB repair. We will also extend these studies to include disease mutations within MR, which will not only allow us to understand how these alterations corrupt MR function and lead to disease but will also provide additional avenues for probing the structures, dynamics, and functional relationships within this complex. Our long-term goals seek to move beyond the core MR complex. Although the MR studies proposed herein will be performed on the simplified construct of Rad50, which has been used in all previous x-ray crystallographic studies of MR, we aim to perform, for the first time, similar studies using full-length Rad50. In total, our research program aims to better understand how macromolecular assemblies use protein motions to regulate their functions, and in the process of applying our program to MRN, we will determine the effect that large and small scale motions have in controlling the first steps in MRN-mediated DNA DSB repair.