engleski

The role of Tyrosyl-DNA-phosphodiesterases in the repair of DNA-protein crosslinks in vivo

DNA protein crosslink (DPC) is a type of DNA lesion which occurs when protein becomes irreversibly covalently bound to DNA. These lesions are caused by exposure to endogenous and/or exogenous crosslink inducers. If not repaired, DPCs interfere with all DNA transactions, thus causing genomic instability which can lead to cancer, accelerated aging and neurodegeneration. The orchestration of DPC repair pathway is still unknown, especially in vivo. Recently, it has been shown that metalloproteases Wss1 in yeast and SPRTN in mammals play central role in the repair of DPCs. These proteases cleave crosslinked proteins, thus leaving protein remnants of unknown size in the DNA backbone. While proteases act ubiquitously, cleaving wide variety of general DPCs, different set of proteins are involved in the repair of enzymatic DPC. Enzymatic DPCs are formed when proteins which are reversibly bound to DNA in order to perform their function become crosslinked due to endogenous and/or exogenous inducers. The most known enzymatic DPCs include topoisomerase 1 and 2 which are also known as topoisomerases 1 and 2 cleavage complexes. Tyrosyl phosphodiesterase 1 and 2 (TDP1 and 2) play crucial role in the removal of these enzymatic DPCs. TDP1 has been shown to remove protein remnant of TOPO1cc by separating DPC from DNA backbone through esterase activity, most probably after SPRTN mediated proteolytic cleavage of TOPO1 crosslinked to DNA. TDP2 can act (a) downstream of SPRTN mediated proteolysis of TOPO2 DPC or (b) together with ZATT protein to remove TOP2 DPC independent of SPRTN proteolysis. Understanding this type of repair is crucial because of the wide range of chemotherapeutic that are used specifically to induce enzymatic DPCs, primarily TOPO1 -ccs and TOPO2-ccs. Such drugs include camptothecin and its derivatives that induce TOP1 -ccs and etoposide which induces TOPO2-ccs. These drugs are used in a wide range of cancer treatments. We aim to show the function of TDPs in DPC removal in vivo using the zebrafish model using CRISPR/Cas9 mediated mutagenesis of the TDPs active site via knock-in technology and fluorescent reporter. We have identified zebrafish TDPs and compared them to human orthologs in regard to phylogenetics, synteny and mRNA and protein expression across different tissues, while functional studies are on the way. Our study will reveal actual contribution of TDP1 and 2 in the DPC repair pathway at the organismal level.

TDP1 ; TDP2 ; DNA repair ; SPRTN ; DPC repair

nije evidentirano