Defining the function of histone ADP-ribosylation in DNA repair and genome integrity

DNA不断地暴露在各种诱发DNA损伤的物质中，导致每个细胞每天成千上万的DNA损伤。因此，一套被称为DNA损伤反应（DDR）的复杂途径在DNA损伤发生时检测到DNA损伤并激活其修复机制。这些途径对我们的健康和幸福至关重要，它们的功能障碍会导致各种临床症状，包括癌症、神经变性、免疫缺陷和早衰。因此，了解细胞如何对DNA损伤作出反应和修复将提供有关这些疾病的潜在原因的信息，更重要的是，了解如何治疗这些疾病。adp -核糖基转移酶（ARTs）是一类检测DNA损伤并将adp -核糖单元连接到损伤部位的蛋白质上以促进DNA修复的酶。这些酶的抑制剂目前正在成功地用于治疗卵巢癌，并有可能治疗与DDR缺陷相关的其他病理。然而，尽管抗逆转录病毒抑制剂在临床上很重要，但我们对这些酶如何调节DNA修复的了解有限。进一步了解这一点将为利用ART抑制剂治疗与DDR功能障碍相关的疾病提供完善的策略，并为ART如何调节包括细胞生长和分化、基因表达和程序性细胞死亡在内的其他关键过程提供范例。针对DNA损伤，art对DNA损伤处的蛋白质进行修饰的定义尤其不明确，而且对其如何调节修复过程的基础了解甚少。这种情况是组蛋白的缩影，组蛋白是将DNA包装到细胞核中的蛋白质。这些蛋白质是已知的抗逆转录病毒疗法的靶标。然而，组蛋白上的位点对DNA损伤的反应以及它如何调节DNA修复仍然未知。这种机制的缺乏部分是由于缺乏一个适当的实验平台，在这个实验平台中，ARTs和组蛋白基因都可以被操纵，以直接测试ARTs对组蛋白上特定位点的修饰如何在细胞背景下调节DNA修复的假设。我们已经确定，这些标准在模式生物盘基ostelium中是唯一满足的，这为在该系统中识别新的DNA修复因子和概念提供了机会，这些因子和概念随后将应用于人类。我们目前的工作是在盘基骨柱中建立一个实验管道，以鉴定细胞中的组蛋白adp -核糖基化位点，并通过基因操纵组蛋白基因来阻止它们的修饰。这项研究的目的是利用这种独特的方法来测试这些修饰如何调节DNA修复。我们将全面编目组蛋白，以及其中的氨基酸残基，这些组蛋白在DNA损伤时被art修饰。然后，我们将利用盘基骨菌的遗传易感性来破坏特定的组蛋白adp -核糖基化事件，以确定它们在调节DNA修复中的重要性。这将提供一个强大的实验平台，以鉴定新的修复蛋白，这些修复蛋白被募集到盘基骨菌的adp核糖基化组蛋白中。在确定了这些因素之后，我们将随后描述等效蛋白质如何调节人类的DNA修复。除了进一步了解细胞如何促进DNA修复以防止突变外，这些研究还将提供信息，以促进设计针对DNA修复途径的特定治疗剂，以治疗包括癌症在内的各种疾病。

DNA is continually being exposed to a variety of agents that induce DNA damage resulting in tens-of-thousands of DNA lesions per cell every day. As such, an intricate set of pathways known as the DNA damage response (DDR) detect DNA damage when it occurs and activate mechanisms for its repair. These pathways are critical for our health and well-being and their dysfunction can lead to a variety of clinical symptoms including cancer, neurodegeneration, immune-deficiencies and premature ageing. Therefore, understanding how cells respond to and repair DNA damage will provide information about the underlying causes of these conditions and, importantly, how they can be treated. This strategy is exemplified by inhibition of ADP-ribosyltransferases (ARTs), a class of enzymes that detect DNA damage and attach ADP-ribose units onto proteins at damage sites to promote DNA repair. Inhibitors of these enzymes are currently being used successfully to treat ovarian cancer and have the potential to treat other pathologies associated with defects in the DDR. However, despite the importance of ART inhibitors in the clinic, our knowledge of how these enzymes regulate DNA repair is limited. Furthering this understanding will underpin refined strategies that exploit ART inhibitors to treat diseases associated with DDR dysfunction and provide a paradigm for how ARTs regulate other critical processes including cell growth and differentiation, gene expression and programmed cell death.The proteins modified at DNA lesions by ARTs in response to DNA damage are particularly ill-defined and the basis of how this regulates the repair process is only poorly understood. This situation is epitomized by histones, the proteins that package DNA into the nucleus of the cell. These proteins are known targets for ARTs. However, the sites modified on histones in response to DNA damage and how this regulates DNA repair remains unknown. This lack of mechanistic insight is due, in part, to the absence of an appropriate experimental platform in which both ARTs and histone genes can be manipulated to directly test hypotheses of how modification of specific sites on histones by ARTs regulates DNA repair in a cellular context. We have established that these criteria are uniquely met in the model organism Dictyostelium, providing the opportunity to identify novel DNA repair factors and concepts in this system that will subsequently be applied to humans.Our current work has developed an experimental pipeline in Dictyostelium to identify histone ADP-ribosylation sites in the cell and to genetically manipulate histone genes to block their modification. The aim of this research is to exploit this unique approach to test how these modifications regulate DNA repair. We will comprehensively catalogue the histones, and the amino acid residues in them, that are modified by ARTs in response to DNA damage. We will then exploit the genetic tractability of Dictyostelium to disrupt the specific histone ADP-ribosylation events identified to establish their importance in regulating DNA repair. This will provide a robust experimental platform to identify novel repair proteins that are recruited to ADP-ribosylated histones in Dictyostelium. Having identified these factors, we will subsequently characterize how the equivalent proteins regulate DNA repair in the humans. In addition to providing an increased understanding of how cells promote DNA repair to prevent mutagenesis, these studies will provide information to facilitate the design of specific therapeutic agents to target DNA repair pathways to treat a variety of diseases including cancer.