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Identification of DNA-Protein-Crosslink Repair Factors

Periodic Reporting for period 1 - DNAProteinCrossRep (Identification of DNA-Protein-Crosslink Repair Factors)

Reporting period: 2018-06-01 to 2020-05-31

All living organisms are constantly challenged by many different sources that damage the DNA. It is essential for the individual’s health but also for the transmission of genetic information throughout generations that damages are recognized and faithfully repaired. Inadequate repair leads inevitably to mutations and/or cell death. These are the underlying cause for aging in general but also many severe and unfortunately widespread diseases such as cancer and neurodegeneration. The cell’s response to a distinct type of damage is highly specific and tightly regulated. By now, most repair pathways are relatively well described but how cells respond to DNA-protein crosslinks (DPCs) is less well established. DPCs are formed when either DNA-modifying enzymes get trapped on the DNA while they execute their function or when reactive agents unspecifically crosslink proteins to DNA. In any case, DPCs are large structures that block vital DNA related processes like DNA transcription or replication, and thus are deleterious for the cell, if left unrepaired. It is known that upon DPC recognition, the protein part is removed and degraded, leaving the DNA with a smaller lesion, which subsequently is removed by well-known repair pathways. The protease DVC1 (SPRTN, SPRTN-1 in C. elegans or Wss1 in yeast) was established as a main player of the pathway. The protein is targeting to the DPC via a ubiquitin signal and breaks down the crosslinked protein allowing for downstream processing. It is largely unknown whether alternative factors exist that act similar to DVC1 and how the pathway is regulated. The main objective of this project was to identify and characterize new factors of the DPC repair pathway. I wanted to combine the human cell culture system, a great tool to perform genome wide screens and to study molecular processes, with the power of an in vivo system, the nematode C. elegans. Studying protein function in the worm would allow me to conduct various dedicated studies for newly identified DPC repair factors, e.g. in proliferating and non-proliferating tissues, at the various developmental stages as well as assess the impact on overall organismal fitness and hereby advance my research on the newly identified factors in a physiological relevant context.
We performed a genome wide screen for DPC repair factors and could identify candidates that function in the dedicated repair pathway. After initial verification experiments of candidates, we were able to characterize the function of one of the candidate proteins in detail and show its recruitment to the DPCs in a SUMO dependent manner and that its function is required for worm survival after induction of DPCs. Work on a second candidate is still ongoing.
The underlying reason for the knowledge gap concerning the DPC repair pathway can partly be explained by the lack of appropriate tools to study this pathway, especially the possibility of inducing targeted DPCs was lacking. So far only reagents inducing DPCs amongst a variety of different other DNA lesions were available, making research on DPC specific pathways difficult. In our lab, we developed a cell-based system to induce defined DPCs. We used this system to perform genome wide screens that would allow us identifying genes important for DPC repair. A thorough process of optimisation enabled us to identify several potential candidates. After initial verification with specific siRNAs targeting those candidate genes and establishment of various tools to study their molecular functions, we are currently focusing on one very promising candidate. In-depth molecular characterization is now ongoing. Simultaneously to the screen, another candidate gene was identified by extensive literature and domain structure analysis. We characterized its function in DPC repair by means of high-content microscopy to monitor the repair capacity of different mutants as well as by assessing the survival of those mutants, identifying and analysing specific protein protein interactions and following the recruitment of candidate protein to lesion sites. We used the C. elegans system to unravel the physiological relevance of the newly identified gene, mostly by survival studies. These experiments indicate that the candidate protein is recruited to SUMOylated DPCs, which is in contrast to the recruitment of DVC1, the known DPC repair protein that requires ubiquitylation of DPCs. Furthermore, it protects against DPC toxicity in the nematode C. elegans. Concluding, we provided important, clinically relevant advances in our understanding on how cells and organisms tackle the harmful effects posed by DPCs. I made an effort to exploit and disseminate to the scientific community: I communicated our findings to colleagues in talks, posters and associated discussion at diverse occasions such as international conference and on-site meetings. We have published one article that is available open access, so that everyone can follow our progress. We have a second publication with a follow-up work in revision, that will also be published open access. I presented our work in scope of the EU-funded ENABLE symposium that also included public outreach events to make exciting ongoing science accessible to a broader audience.
Our DNA is constantly challenged by many exogenous but also endogenous genotoxic insults. Different highly elaborate repair mechanisms have developed to counteract these deleterious incidents. If DNA damage gets overwhelming and/or repair pathways are erroneous or malfunctioning persistent DNA damages are inevitable and lead to genomic instability and/or cell death, the underlying cause of many pathologies, including cancer. Hence, faithful repair is a prerequisite of maintaining health and prevent development of diseases. Obviously, it is of major importance to understand how the integrity of DNA is preserved: On the one hand to understand aging in general, but on the other hand to also delineate disease development and discover new and refined treatment strategies for otherwise deleterious diseases. Research over many years has achieved a lot and much is known about players and mechanisms of dedicated repair mechanism and their interplay. Still, there are quite some knowledge gaps, that need to be addressed urgently. One of them is how the deleterious DNA damage posed by DPCs is repaired. Understanding DPC repair in significant detail not only allows to describe how the lack of DPC repair can be a driver of tumorigenesis, but it also gains special importance knowing that DPC inducing agents are widely used in treatment protocols for a variety of malignancies. Hence, it can only be beneficial to understand how these types of lesions are handled by cells: not only to understand the mode of action of different therapeutic drugs and the possible side effects they induce, but also to identify new druggable targets and creating new entry points for possible future therapies, all of this feeding into a better patient care.
SUMO-dependent resolution of DPCs