Regulation of DNA repair by histone ADP-ribosylation

DNA不断地暴露在各种导致DNA损伤的试剂中。因此，一系列被称为DNA损伤反应(DDR)的通路在DNA损伤发生时检测并激活修复机制。这些通路对我们的健康至关重要，它们的功能障碍会导致各种病理现象，包括癌症风险增加、神经变性、先天性异常和过早衰老。因此，了解细胞如何修复DNA损伤将提供关于这些疾病的潜在原因的信息，以及重要的是，如何治疗这些疾病。这一策略的例子是抑制多(ADP-核糖)聚合酶(PAPS)，这是一类通过ADP-核糖化过程将ADP-核糖单位连接到蛋白质上来促进DNA修复的酶。PAPS的抑制剂正被用于治疗乳腺癌和卵巢癌，并有可能治疗与DDR缺陷相关的其他病理疾病。然而，尽管它们有潜在的治疗靶点，但我们对PAPS如何调控DNA修复的了解有限。例如，ADP核糖化的蛋白质对DNA损伤的反应以及这如何调节修复的定义并不明确。这种情况以组蛋白为代表，组蛋白是将DNA包装到细胞核中的蛋白质。组蛋白是PAPS的主要靶点，特别是丝氨酸氨基酸，它也有可能被磷酸化修饰。由于磷酸化调控包括细胞生长和分裂在内的各种过程，这增加了ADP-核糖化和磷酸化之间的相互作用可能通过各种途径协调DNA修复的可能性。然而，由于在人类细胞中操纵组蛋白基因的困难，这些关系的功能意义仍有待测试。因此，需要一个组蛋白基因可以很容易地操纵组蛋白基因的实验系统来测试组蛋白ADP-核糖化如何调控DNA修复。我们目前由MRC资助的工作为这些问题提供了关键进展，方法是在变形虫Dictyostelial中建立一个强大的实验管道，使我们能够操纵组蛋白基因，以评估组蛋白ADP-核糖化如何调控DNA修复。我们以前的工作率先使用该系统来研究在其他遗传模式生物中丢失的人类DNA修复机制，包括ADP-核糖化。通过利用这种生物体中独特的操纵组蛋白ADP-核糖化位点的能力，我们发现组蛋白ADP-核糖化和磷酸化之间的相互作用是通过协调DNA修复和细胞分裂来维持基因组完整性的关键。这为ADP-核糖化如何与其他翻译后修饰整合以调节DDR提供了范式转变，并提供了识别可扩展到人类细胞的新调控机制的能力。拟议的工作将建立在这些关键的技术和概念上的进展，以确定组蛋白ADP-核糖化如何将DNA修复与细胞周期进展结合起来，并将这些发现扩展到人类细胞。除了增加对细胞如何促进DNA修复以防止突变的理解外，这些研究还将提供信息，以促进针对DNA修复途径的治疗剂的设计，以治疗与DDR缺陷相关的病理。

DNA is continually being exposed to a variety of agents that induce DNA damage. As such, a 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 their dysfunction leads to a variety of pathologies including increased cancer risk, neurodegeneration, congenital abnormalities and premature ageing. Therefore, understanding how cells 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 Poly(ADP-ribose)-polymerases (PARPs), a class of enzymes that promote DNA repair by attaching ADP-ribose units onto proteins through a process known as ADP-ribosylation. Inhibitors of PARPs are being used to treat breast and ovarian cancers and have the potential to treat other pathologies associated with DDR defects. However, despite their potential as therapeutic targets, our knowledge of how PARPs regulate DNA repair is limited. For example, the proteins ADP-ribosylated in response to DNA damage and how this regulates repair are ill-defined. This situation is epitomized by histones, the proteins that package DNA into the nucleus of the cell. Histones are major targets for PARPs, particularly at serine amino acids that also have the potential to be modified by phosphorylation. Given phosphorylation regulates a variety of processes, including cell growth and division, this raises the possibility that interplay between ADP-ribosylation and phosphorylation may coordinate DNA repair with a variety of pathways. However, the functional significance of these relationships remains to be tested due to difficulties in manipulating histone genes in human cells. There is therefore a need for an experimental system where histone genes can be easily manipulated to test how histone ADP-ribosylation regulates DNA repair.Our current MRC-funded work provided key advances to these questions by developing a robust experimental pipeline in the amoeba Dictyostelium that allowed us to manipulate histone genes to assess how histone ADP-ribosylation regulates DNA repair. Our previous work pioneered the use of this system to study human DNA repair mechanisms lost in other genetic model organisms, including ADP-ribosylation. By exploiting the unique ability to manipulate histone ADP-ribosylation sites in this organism, we identified that interplay between histone ADP-ribosylation and phosphorylation is critical to maintain genome integrity by coordinating DNA repair with cell division. This provides a paradigm shift for how ADP-ribosylation integrates with other post-translational modifications to regulate the DDR and the ability to identify novel regulatory mechanisms that can be extended to human cells. The proposed work will build on these key technical and conceptual advances in Dictyostelium to identify how histone ADP-ribosylation couples DNA repair with cell cycle progression and extend these findings to human cells. 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 therapeutic agents that target DNA repair pathways to treat pathologies associated with a defective DDR.