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The role of ubiquitin and ubiquitin-like modifiers in replication stress

Final Report Summary - UBIREPS (The role of ubiquitin and ubiquitin-like modifiers in replication stress)

Genome integrity strictly depends on accurate and faithful transmission of genetic information between generations. Key prerequisite for faithful genome duplication are the elaborate mechanisms regulating DNA replication. These mechanisms ensure that the genome is duplicated accurately and completely before the DNA is segregated and distributed equally to the daughter cells during the ensuing cell division. The importance of these mechanisms for human health is best illustrated by the fact that the perturbation of DNA replication, a phenomenon known as “replication stress”, is a key source of genome instability and a principal driver of tumor development. Replication stress can result from endogenous sources, e.g. genetic defects or reactive cellular metabolites, and from environmental influences, e.g. UV irradiation or chemotherapeutic treatment. While nature has evolved efficient means to overcome these obstacles to DNA replication, some of them invariably escape these repair activities and cause mutations that promote disease.
The aim of the UBIREPS project is to identify novel components and pathways that limit the deleterious consequences of DNA replication perturbation. To this end, we established an assay that identifies genes specifically required for efficient DNA replication under conditions of stress. Our assay combines quantitative high-content microscopy with reverse genetics, provides us with multi-parameter single cell data, and is suitable for large-scale screens of hundreds or thousands of genes. We have employed the assay to sample a library of approximately 1400 human genes. Our screen has identified a number of genes as potential regulators of the DNA replication stress response. After validation by independent assays, we are in the process of elucidating the mechanisms by which these genes contribute to maintain genome stability in face of replication stress.
In order to compliment the screening approach, we are investigating in parallel a second, closely related aspect of DNA replication stress, i.e. the consequences of perturbed DNA replication for the ensuing cell generations. Failure to fully resolve persistent obstacles to DNA replication is known to impair completion of DNA replication. Incompletely replicated sections of the genome interfere with segregation during cell division, and thus pose a major threat to genome integrity. In order to minimize these hazards to genome integrity, cells have evolved specialized processing machineries that cleave the incompletely replicated DNA and allow distribution of the DNA into the daughter cells. However, processing comes at the price of DNA lesions in these daughter cells. While the formation of these so-called “53BP1 nuclear bodies” is reasonably well understood, the fate of 53BP1 nuclear bodies in proliferating cells is completely enigmatic. Our experiments revealed when these lesions are repaired, and we are currently deciphering the molecular details and regulation of the processes that are required for successful repair.
Together, our studies identify hitherto unknown mechanisms protecting genome integrity, both in immediate and in delayed responses to replication perturbation. We describe how malfunctioning of these mechanisms impacts on human health and disease, which will not only advance our understanding of the intricate workings of the cell, but also point towards novel avenues for cancer treatment. While on-going clinical trials are revealing the potential of targeting the DNA replication for cancer therapy, mechanistic insight into the new facets of replication stress responses is key to rationalize predictions of current treatment outcomes and to identify novel drugable targets.