Mechanism and Regulation of Human Nonhomologous DNA End Joining

摘要 大多数人类癌症都有染色体结构变化，双链DNA断裂 (DSB)是主要原因。非同源DNA末端连接(NHEJ)是修复的主要途径 DSB和NHEJ具有足够的灵活性，可以连接任何一对DNA末端，无论它们的结构如何。 NHEJ的灵活性是至关重要的，因为DSB的自然原因(例如电离辐射、反应性 氧物种，失败的核酶反应)产生具有不同分子的DNA末端 配置。因此，这种灵活性非常适合这项任务，但它会产生负面影响，导致 几乎所有修复部位的DNA序列改变。NHEJ代表了最复杂的 蛋白质：DNA相互作用途径，因为两者之间暂时不存在共价连接 DNA结束了。尽管我们知道大多数参与NHEJ的蛋白质，而且从广义上讲， 它们是如何发挥作用的，我们不清楚它们的空间和时间相互作用，或者一些 根据DNA末端分子的不同，某些NHEJ事件需要组件，而其他事件则不需要 配置。有了更清晰的人类NHEJ的机制和结构图景，我们将处于 为了开发可用于治疗许多癌症的小分子抑制剂(例如， 化学疗法或作为辐射增敏剂)。目标1描述了我们的创新步骤 阐明Artemis：DNA-PKcs：Ku复合体的结构。虽然在细胞内有一些单独的蛋白质 复合体具有已知的结构(例如，Ku和部分DNA-PKcs)，我们想要了解 NHEJ蛋白需要不同的组装，如Ku，DNA-PKcs和Artemis，以修复 细胞中出现的各种DNA末端分子构型。目标2A和2B描述了我们的能力 使用纯化的蛋白质和我们分析哪些步骤的能力直接遵循NHEJ的化学步骤 对于每种类型的DNA末端配置都是至关重要的。目标2C和2D决定如何包裹DNA 组蛋白八聚体(单核小体)周围的双链影响NHEJ的连接机制和化学 在台阶上。目标3检查过程中两个DNA末端的非共价近似(突触) NHEJ，利用sm-fret对其进行了优化研究。目标3A确定突触的程度 步骤(DNA两端的近似)决定了NHEJ的总体比率。AIM 3B使用sm-fret 检测核小体DNA是否能被NHEJ蛋白突触。目标4描述了我们的创新 DNA分解必需核酸酶Artemis小分子抑制剂的研究进展 修复因电离辐射或拓扑异构酶II抑制剂(两者)造成的损害的中间体 用于治疗)。43.3万种化合物的高通量筛选鉴定了20种化合物 这值得进一步研究，其中一个子集满足高度严格的人类细胞生物选择性测试 特定的一半反应产物被阻断，而不影响另一半或DNA代谢。

ABSTRACT Most human cancers have chromosomal structural changes, and double-strand DNA breaks (DSBs) are the major cause. Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing DSBs, and NHEJ is sufficiently flexible that it can join any pair of DNA ends, regardless of their structure. The flexibility of NHEJ is essential because natural causes of DSBs (e.g., ionizing radiation, reactive oxygen species, failed nuclear enzyme reactions) generate DNA ends with diverse molecular configurations. Hence, that flexibility is well suited for the task, but it has the negative impact of causing DNA sequence alterations at nearly all repair sites. NHEJ represents one of the most sophisticated protein:DNA interaction pathways because transiently there is no covalent connection between the two DNA ends. Although we know most of the proteins that participate in NHEJ and know, in broad terms, how they function, we do not have a clear picture of their spatial and temporal interactions, or how some components are required for some NHEJ events but not others, depending on the DNA end molecular configuration. With a clearer mechanistic and structural picture of human NHEJ, we will be in a position to develop small molecule inhibitors that may be useful for treating many cancers (e.g., chemotherapeutically or as a radiation sensitizer). Aim 1 describes our innovative steps toward elucidating the structure of the Artemis:DNA-PKcs:Ku complex. Though a few individual proteins in the complex have known structures (e.g., Ku and portions of DNA-PKcs), we want to understand how different assemblies of the NHEJ proteins, such as Ku, DNA-PKcs, and Artemis, are needed to repair the various DNA end molecular configurations that arise in the cell. Aims 2A and 2B describe our ability to directly follow the chemical steps of NHEJ using purified proteins and our ability to dissect which steps are critical for each type of DNA end configuration. Aim 2C and 2D determine how wrapping the DNA duplex around histone octamers (mononucleosomes) affects the NHEJ joining mechanism and chemistry of the steps. Aim 3 examines the noncovalent approximation (synapsis) of the two DNA ends during NHEJ, which is optimally studied using sm-FRET. Aim 3A determines the extent to which the synapsis step (approximiation of the two DNA ends) determines the overall rate of NHEJ. Aim 3B uses sm-FRET to test whether nucleosomal DNA can be synapsed by NHEJ proteins. Aim 4 describes our innovative development of small molecule inhibitors of Artemis, which is an essential nuclease for resolving DNA repair intermediates due to damage from ionizing radiation or topoisomerase II inhibitors (both of which are used therapeutically). A high throughput screen of 433,000 compounds has identified 20 compounds that merit further study, and a subset satisfy a highly stringent human cellular bioselectivity test in which a specific half of the reaction products are blocked without affecting the other half or DNA metabolism.