DNA-protein crosslinks: endogenous origins and cellular responses.

Información del proyecto

DNAProteinCrosslinks

Identificador del acuerdo de subvención: 801750

DOI

10.3030/801750

Proyecto cerrado

Fecha de la firma de la CE 24 Octubre 2018

Fecha de inicio 1 Febrero 2019

Fecha de finalización 31 Marzo 2024

Financiado con arreglo a

• EXCELLENT SCIENCE - European Research Council (ERC)

Coste total

€ 1 497 375,00

Aportación de la UE

€ 1 497 375,00

Coordinado por

LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Germany

Periodic Reporting for period 4 - DNAProteinCrosslinks (DNA-protein crosslinks: endogenous origins and cellular responses.)

Período documentado: 2023-08-01 hasta 2024-03-31

Resumen del contexto y de los objetivos generales del proyecto

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.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

Main achievements:

1.) 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.

2.) 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).

3.) We identified helicase-mediated protein unfolding by the FANCJ helicase to be required for replication-coupled DPC repair (Yaneva et al., Mol Cell 2023).

4.) We identified a non-proteolytic release mechanisms for endogenous DPCs formed by the HMCES protein (Donsbach et al., EMBO J 2023).

5.) We successfully developed the proposed new methodology to determine the nature of the protein component of DPCs in various experimental scenarios (Weickert et al., Nat Commun 2023) as well as their genomic position (Carnie et al., Nat Cell Bio 2024). Withs these tools in hand, we have discovered and characterised novel global-genome as well as transcription-coupled DNA repair pathways with imminent relevance for the aetiology of human premature ageing and cancer predisposition syndromes (Weickert et al., Nat Commun 2023, Carnie et al., Nat Cell Bio 2024, Carnie et al., EMBO J 2024).

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

The methodologies developed for the detection and identification of cellular DPCs are a game-changer for the genome stability field and will be applicable to various basic and applied research questions. Moving forward, we will use these new technologies 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 of endogenous DPC formation.

Schematic depiction of different DNA lesions