Regulation of DNA repair by histone ADP-ribosylation

DNA不断暴露于各种诱导DNA损伤的试剂。因此，一组称为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。这些通路对我们的健康至关重要，它们的功能障碍导致各种病理学，包括癌症风险增加，神经退行性变，先天性异常和过早衰老。因此，了解细胞如何修复DNA损伤将提供有关这些疾病的根本原因的信息，更重要的是，如何治疗这些疾病。这种策略的例子是抑制聚（ADP-核糖）-聚合酶（PARP），这是一类通过ADP-核糖基化将ADP-核糖单元连接到蛋白质上来促进DNA修复的酶。PARP的抑制剂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。然而，尽管它们作为治疗靶点的潜力，我们对PARP如何调节DNA修复的知识是有限的。例如，蛋白质ADP核糖基化对DNA损伤的反应以及如何调节修复是不明确的。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。组蛋白是PARP的主要靶点，特别是在丝氨酸氨基酸上，这些氨基酸也有可能被磷酸化修饰。鉴于磷酸化调节多种过程，包括细胞生长和分裂，这增加了ADP-核糖基化和磷酸化之间的相互作用可能协调DNA修复与多种途径的可能性。然而，这些关系的功能意义仍有待测试，由于在人类细胞中操纵组蛋白基因的困难。因此，需要一个实验系统，可以很容易地操纵组蛋白基因来测试组蛋白ADP-核糖基化如何调节DNA修复。我们目前的MRC资助的工作提供了这些问题的关键进展，通过开发一个强大的实验管道，在阿米巴Dictyosteelium，使我们能够操纵组蛋白基因，以评估组蛋白ADP-核糖基化如何调节DNA修复。我们以前的工作开创了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-核糖基化。通过利用这种生物体中操纵组蛋白ADP-核糖基化位点的独特能力，我们确定了组蛋白ADP-核糖基化和磷酸化之间的相互作用对于通过协调DNA修复与细胞分裂来维持基因组完整性至关重要。这为ADP-核糖基化如何与其他翻译后修饰整合以调节DDR以及鉴定可扩展至人类细胞的新型调节机制的能力提供了范式转变。拟议的工作将建立在这些关键的技术和概念上的进展，在Dictyosteoblasts，以确定如何组蛋白ADP-核糖基化夫妇DNA修复与细胞周期进程，并将这些发现扩展到人类细胞。除了提供对细胞如何促进DNA修复以防止诱变的更多理解之外，这些研究将提供信息以促进靶向DNA修复途径的治疗剂的设计，以治疗与缺陷DDR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.