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

点击翻译按钮获取中文摘要

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.