Periodic Reporting for period 3 - DNAProteinCrosslinks (DNA-protein crosslinks: endogenous origins and cellular responses.)
Période du rapport: 2022-02-01 au 2023-07-31
The genetic material - the DNA - in all of our cells is constantly threatened by various types of damage. Sequence and structure of the DNA are subjected to lesions ranging from chemical alterations to breaks of the DNA double helix. How cells, detect, signal and eventually repair these lesions has been studied for many decades. Surprisingly however, how cells detect one particular type of lesion - covalent DNA-protein crosslinks (DPCs) - had remained enigmatic, until the recent identification of specific enzymes - so-called DPC protease - targeting crosslinked protein adducts directly. These discoveries brought DPCs into the focus of genome stability research. Strikingly, the human DPC protease SPRTN is required for viability of human cells suggesting that cells constantly face substantial amounts of endogenous DPCs. The fundamental importance of DPC repair by SPRTN is further underlined by the premature aging and cancer predisposition observed in patients bearing partial-loss-of-function mutations in SPRTN. However, the cellular processes causing endogenous DPC formation remain largely unclear. Moreover, many widely-used chemotherapeutic agents exert their therapeutic potential by inducing DPCs. Nonetheless, how cells detect and repair these lesions is only poorly understood. Addressing these pressing questions has remained challenging, due to limitations of the currently available methodology to study DPCs and their repair.
The Project DNAProteinCrosslinks set out to close this knowledge gap by attempting to reveal the identity and sources of endogenous DPCs as well as to determine the cellular responses to these threats in mechanistic detail. To achieve this global objective, we pursued the following three aims: (1) Identification of factors and signals regulating protease-based DPC repair, (2) achieving a system-wide view of DPCs and their repair by developing a novel broadly applicable method to overcome current technical limitations to study DPCs and their repair in a global system-wide manner, and (3) revealing the origins of endogenous DPCs by conducing genetic screens to identify the cellular processes causing lethality in the absence of SPRTN.
The Project DNAProteinCrosslinks set out to close this knowledge gap by attempting to reveal the identity and sources of endogenous DPCs as well as to determine the cellular responses to these threats in mechanistic detail. To achieve this global objective, we pursued the following three aims: (1) Identification of factors and signals regulating protease-based DPC repair, (2) achieving a system-wide view of DPCs and their repair by developing a novel broadly applicable method to overcome current technical limitations to study DPCs and their repair in a global system-wide manner, and (3) revealing the origins of endogenous DPCs by conducing genetic screens to identify the cellular processes causing lethality in the absence of SPRTN.
In this reporting period, we made substantial progress on all aims. We conducted biochemical screens to reveal the first factors controlling the activity of the SPRTN protease in human cells (Zhao et al., NAR 2021). We revealed that the enzyme USP7 governs a regulatory switch, which controls SPRTN by switching the enzymes preference from inactivating autocleavage to substrate cleavage in times of need. Moreover, we revealed how the activity of SPRTN is restricted to crosslinked protein adducts, while leaving surrounding chromatin proteins unharmed. By developing new model substrates, we were able to show that SPRTN achieves specificity by recognizing specific DNA structures, which restricts activation of the enzyme to biologically relevant scenarios (Reinking et al., Mol Cell 2020, Reinking and Stingele, STARProtocols 2021).
We successfully developed the proposed new methodology to detect DPCs in various experimental scenarios (Weickert, Li et al., in preparation). To enable the proposed genetic screens, we developed various tools and model systems including inducible human SPRTN knock-out cells and CRISPR-engineered hypomorphic SPRTN mutant cells mimicking disease-causing patient variants.
We successfully developed the proposed new methodology to detect DPCs in various experimental scenarios (Weickert, Li et al., in preparation). To enable the proposed genetic screens, we developed various tools and model systems including inducible human SPRTN knock-out cells and CRISPR-engineered hypomorphic SPRTN mutant cells mimicking disease-causing patient variants.
The methodology developed for the detection and identification of cellular DPCs is a game-changer for the genome stability field and will be applicable to various basic and applied research questions. Moving forward, we will use this new technology to study the currently elusive cellular principles governing DPC repair pathway choice. Moreover, we will employ it to determine the identity and nature of endogenous DPCs. In combination with our genetic approaches this will bring us in the position to settle the question of the origins if endogenous DPC formation.