engleski

THE ROLE OF TYROSIL PHOSPHODIESTERASE 2 IN DPC REPAIR in vivo

DNA-protein crosslinks (DPCs) are formed when a protein irreversibly covalently binds to DNA, physically disrupting all DNA transactions. DPCs cause genomic instability that can lead to cancer, accelerated aging, and neurodegeneration. These lesions are caused by different proteins, so it is no coincidence that cells have evolved multiple repair pathways. One of the most common cellular DPCs is Topoisomerase 2 (TOPO2), an enzyme important for generating double-strand breaks to reduce the torsional stress on DNA during replication, transcription and chromatin remodeling. Several drugs such as etoposide have been developed to stimulate the formation of Topoisomerase 2 DPCs (TOPO2- DPCs), leading to cancer cell death. TOPO2-DPCs can be repaired (1) by SPRTN protease and Tyrosyl phosphodiesterase 2 (TDP2), (2) by MRE11 nuclease during DNA end resection, and (3) by the synchronized action of zinc finger protein ZATT and TDP2. To date, the mechanisms of TOPO2-DPCs repair are partially unknown, whereas their repair at the organism level has not been investigated. Therefore, we set out to determine the role of TDP2 in TOP2-DPCs repair in to the context of SPRTN protease and MRE11 nuclease, which have previously been implicated in repair in vitro. Mutations of TDP2 in humans have been associated with intellectual disability, seizures, and spinocerebellar ataxia. To investigate the role of TDP2 protein in DPC repair at the organismal level, we decided to generate zebrafish strains lacking TDP2 protein and strains with enzymatically inactive protein. Since zebrafish has two orthologs of the TDP2 protein, Tdp2a and Tdp2b, we used CRISPR/Cas genome editing to mutate both genes. We successfully introduced a premature STOP codon into the Tdp2a protein and were able to delete the enzymatically important Glu at position 232. For the Tdp2b protein, we created a fish line into which we introduced the STOP codon and a strain carrying a point mutation at position A4860C that produces an enzymatically impaired Tdp2b. These animal models will reveal (1) which TOPO2 repair pathway is predominant in the organism and (2) whether TDP2 can help eliminate other DPCs. In addition, our study will help to better understand diseases associated with TDP2 deficiency.

TDP2, DPC, SPRTN, Topoisomerase 2,

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