ESR6: “Regulation of DNA-protein crosslink repair in human cells”

UCPH (N. Mailand group): Regulation of DNA-protein crosslink repair in human cells

i. Objective of research: To delineate the regulatory mechanisms by which cells respond to and overcome cytotoxic DNA-protein crosslinks.

ii. Current state of the art: DNA-protein crosslinks (DPCs) are formed when DNA-modifying enzymes become trapped during catalysis or by a variety of endogenous and exogenous agents that non-specifically crosslink proteins in the vicinity of DNA. Due to their large size, DPCs obstruct essential DNA-associated processes such as DNA replication and transcription, and the ability of cells to remove these lesions is therefore critical for cell and organismal fitness. However, while the repair systems for most types of DNA damage are now relatively well understood, the mechanisms by which cells respond to DPCs remain profoundly underexplored, largely due to a lack of efficient approaches for inducing defined DPCs into the genome. Recent studies in yeast and vertebrate cells revealed the existence of a dedicated protease-based DPC repair pathway. However, whether additional DPC repair factors exist and how DPC recognition and repair processes are orchestrated and regulated remain to be established. As many commonly used chemotherapeutic agents are potent DPC inducers, a detailed understanding of the mechanisms underlying DPC recognition and removal is of paramount biomedical importance, and will help to foster an improved understanding of current cancer treatment regimens and the rational development of more targeted and efficacious therapeutic strategies.

iii. Research methodology and approach: Using a simple yet robust strategy for inducing controlled DPCs in human cells, we found that DPC formation triggers strong SUMO-dependent modification of the covalently crosslinked protein and associated factors. Based on our preliminary results, we posit that SUMO plays a crucial role in mitigating the cytotoxicity of DPCs by marking them for recognition and processing by cellular DPC repair factors. To understand how SUMO-dependent signaling promotes DPC repair, we will use state-of-the-art proteomic approaches to perform detailed mapping studies of cellular proteins undergoing DPC-induced SUMOylation. Because DNA damage-dependent SUMOylation responses typically proceed according to a group modification scheme in which many proteins recruited to damaged DNA are simultaneously SUMOylated, these proteomic analyses are expected to reveal critical components of the DPC repair and processing machinery. The potential roles in DPC repair and genome stability maintenance of selected factors undergoing DPC-induced SUMOylation will subsequently be investigated in detail using a wide panel of functional cell-, biochemistry- and advanced imaging assays

iv. Originality and innovative aspects of the ESR project: Using systems-wide and focused approaches, this project will provide important first insights into the regulation of responses by which human cells overcome defined DPCs to safeguard genetic integrity, cell survival and organismal fitness.

v. Integration of the ESR project to the overall research programme: Our ESR will work with the Lingner group on the refinement of mass spectrometry methodologies, with Genevia on novel mass spectrometry analysis pipelines and with the LXRepair and Norgenotech on the development of novel assays for the detection of UV- and IR-induced DNA lesions, respectively.