DNA-protein crosslinks (DPCs) are frequent DNA lesions caused by irreversible covalent bonds between proteins and DNA. These bulky lesions hinder all DNA transactions, and therefore the consequences of impaired DNA-Protein Crosslink Repair (DPCR) are severe. At the cellular level, defective repair causes DNA breaks, genomic instability, and cell death, while at the organismal level, impaired DPCR is associated with cancer, aging, and neurodegeneration. Topoisomerases (TOPs) are among the most abundant DPCs in cells under physiological conditions and TOP-DPC inducers such as topotecan and etoposide are used to treat various cancers. Tyrosyl-DNA phosphodiesterases (TDPs) are enzymes that remove crosslinked topoisomerase peptide residues from the DNA and their deficiency leads to neurological defects in mice and humans. Despite many efforts, the repair of these highly toxic DNA lesions is still not fully understood. We will investigate (1) which proteins are crucial for the repair of TOP1- and TOP2-DPCs in vivo and whether they are epistatic, (2) link between TOP-DPCR and the neurological defects in TDP-deficient organism, and (3) whether TOP-DPCs accumulate in the aging organism. Our approach includes: (a) gene silencing of TOP-DPC repair factors: sprtn, acrc, zatt, tex264 apex1, 2 and mre11 and inhibition of ATPase p97 and proteasome in WT and Tdp-deficient zebrafish embryos, (b) generation of tdp1 and tdp2 double mutant zebrafish strains, (c) characterization of DPC profiles in different backgrounds of gene manipulation in embryos and in different tissues during aging, and (d) identification of neurological changes including ataxia, anxiety, learning and memory in Tdp1- and Tdp2-deficient adult fish. The proposed project will be the first to investigate TOP-DPC repair at the organismal level. It will improve our understanding of human disease and aging and could lead to the identification of new drug targets for the treatment of cancer and neurological diseases.