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

DNA不断暴露于各种诱导DNA损伤的试剂中，导致每天每个细胞成千上万的DNA损伤。因此，一组复杂的称为DNA损伤反应（DDR）的途径在DNA损伤发生时检测DNA损伤并激活其修复机制。这些途径对我们的健康和福祉至关重要，它们的功能障碍可能导致各种临床症状，包括癌症，神经退行性变，免疫缺陷和过早衰老。因此，了解细胞如何应对和修复DNA损伤将提供有关这些疾病的根本原因的信息，重要的是，如何治疗它们。这种策略的例子是抑制ADP-核糖基转移酶（ART），这是一类检测DNA损伤并将ADP-核糖单元连接到损伤位点处的蛋白质上以促进DNA修复的酶。这些酶的抑制剂目前被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ART抑制剂在临床上很重要，但我们对这些酶如何调节DNA修复的知识是有限的。进一步理解这一点将支持利用ART抑制剂治疗与DDR功能障碍相关的疾病的精细策略，并为ART如何调节其他关键过程（包括细胞生长和分化）提供范例，基因表达和程序性细胞死亡。在DNA损伤处被ART修饰的蛋白质对DNA损伤的反应特别不良，定义和基础，这是如何调节修复过程只是知之甚少。这种情况集中体现在组蛋白上，组蛋白是将DNA包装到细胞核中的蛋白质。这些蛋白质是ART的已知靶点。然而，组蛋白上响应DNA损伤的修饰位点以及这如何调节DNA修复仍然未知。这种缺乏机制的见解，部分是由于缺乏一个适当的实验平台，其中ART和组蛋白基因可以被操纵，以直接测试的假设，如何修改组蛋白上的特定位点的ART调节DNA修复在细胞的情况下。我们已经确定，这些标准是唯一符合模式生物Dictyosteopathy，提供了机会，以确定新的DNA修复因子和概念，在这个系统中，随后将被应用于human.We目前的工作已经开发了一个实验管道Dictyosteopathy，以确定组蛋白ADP-核糖基化位点的细胞和遗传操纵组蛋白基因，以阻止其修改。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。我们将全面地对组蛋白和其中的氨基酸残基进行分类，这些组蛋白和氨基酸残基被ART修饰以应对DNA损伤。然后，我们将利用网柄藻的遗传易处理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，以确定新的修复蛋白，在网骨藻中招募ADP-核糖基化组蛋白。在确定了这些因素之后，我们将随后描述等价蛋白质如何调节人类的DNA修复。除了提供对细胞如何促进DNA修复以防止诱变的更多理解外，这些研究还将提供信息以促进特定治疗剂的设计，以靶向DNA修复途径来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.