DNA-protein crosslinks (DPCs) are DNA lesions with adverse effects on the organismal level, including cancer, premature aging and neurodegenerative diseases. DPCs occur when a protein irreversibly covalently binds to DNA and thus impair all DNA transactions: replication, transcription and repair. DPC repair (DPCR) is therefore an essential cellular pathway which was overlooked due to the fact that it was not considered to be a distinct DNA damage repair pathway until recent discoveries of proteases Wss1 and SPRTN with central role in the pathway. However, molecular mechanisms and structural knowledge behind the protease-mediated DPCR is lacking. To date, structural information on DPCs and DPCR factors is limited to a fragment containing the active site of SPRTN protease. Data from yeast indicate that SPRTN might work in concert with the ATP-dependent AAA family segregase p97, another essential protein linked to DPCR. In addition, we have recently found another SPRTN-like putative protease, ACRC (syn. GCNA) shown to be linked to DPCR. Currently, structural data on SPRTN interaction with DPC substrates and p97 segregase is lacking, as well as biochemical and structural data on ACRC. We aim to (1) characterize SPRTN:p97 complex and putative protease ACRC in vitro and in vivo, and (2) solve structures of human SPRTN:DPC and SPRTN:p97 complexes as well as the structure of ACRC protein. SPRTN complexes with a model DPC and a p97 will be reconstituted in vitro. Structures of high resolution will be obtained using a state-of-the-art cryo-EM facility with phase plate and a direct electron detector, while the structure of ACRC will be solved using X-ray crystallography due to the small size of the protein. The proposed research will unravel the mechanisms underlying DPCR on a molecular level and will contribute to the field of p97 and SPRTN inhibitor development for the purpose of cancer treatment and modulation of aging processes.