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。我们对理解大分子中的这种关系特别感兴趣。 组件，在过去一直不愿进行详细的结构和动力学研究。我们的 策略是将高分辨率的基于甲基的核磁共振(核磁共振)谱结合在一起， 它能够探测蛋白质及其复合体，大小可达~1丙二醛，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守和 必需DNA双链断裂(DSB)修复复合体Mre11-Rad50-Nbs1(MRN)。这种蛋白质 复合体是检测DNA双链断裂并启动其修复过程的核心。几种疾病 已经注意到MRN的突变，它会导致免疫缺陷、发育和 神经退行性疾病，以及某些癌症的易感性。Mre11基因的其他零星突变 和RAD50已经在许多不同的癌症中被发现。细菌和古生物模型系统， 其中缺乏Nbs1，一直是现有身体X射线结晶学和生化研究的重点 研究表明，蛋白质运动在编排mre11的各种功能中起到了作用- RAD50(MR)核心复合体。然而，关于蛋白质结构和蛋白质之间的相互作用，仍然存在许多问题。 运动以及这些运动与MR活动的关系和控制。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与模仿不同类型的底物DNA完成 DNA双链断裂，并描述发生在这些复合体中的蛋白质动力学。基于核磁共振的 研究将与各种体外生物物理和生化技术相辅相成，以进一步 探测结构域的运动和广泛的MR活性，以及在酵母中的体内研究 这些动作和活动纳入了DNA DSB整体修复的背景下。我们也会扩大这些研究的范围。 将疾病突变包括在MR中，这不仅可以让我们了解这些变化是如何 破坏MR功能并导致疾病，但也将提供用于探测结构的附加途径， 动态，以及这个综合体内的功能关系。我们的长期目标是寻求超越 核心MR复合体。尽管在此建议的MR研究将在简化结构上执行 在所有以前的MR X射线结晶学研究中都使用过的Rad50，我们的目标是为 第一次，类似的研究使用了全长RAD50。总而言之，我们的研究计划旨在更好地 了解大分子组件如何使用蛋白质运动来调节其功能，以及在 将我们的程序应用于MRN的过程中，我们将确定大尺度和小尺度运动的效果 在MRN介导的DNA DSB修复的第一步中具有控制作用。

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.