engleski

Role of tyrosyl DNA-phosphodiesterase 1 in the repair of the DNA-protein crosslinks in vivo

Tyrosyl DNA-phosphodiesterase 1 (TDP1) is a protein that primarily repairs trapped topoisomerase 1 on the DNA backbone. Topoisomerase 1 (TOPO1) is an essential enzyme that relaxes the DNA helix during replication and transcription by introducing a temporary single-strand break in DNA. This reaction is reversible: TOPO1 introduces a single-strand break and afterwards dissolves from the DNA backbone followed by repair of the single- strand break. Due to various endogenous and exogenous factors acting on the cell, TOPO1 can remain irreversibly covalently linked to the DNA backbone thus interfering with all DNA transactions. For this reason, cells have TDP1 protein, which through its phosphodiesterase activity destroys the covalent bond formed between tyrosines in TOPO1 protein and 5 'phosphates on DNA. Lack of TDP1 protein activity leads to spinocerebellar ataxia with axonal neuropathy (SCAN1 syndrome). A large number of drugs for the tumor treatments are aimed at inhibiting the activity of TOPO1 enzyme and/or the formation of TOPO1-DNA irreversible covalent bond. Therefore, understanding of this mechanism is crucial for further development of chemotherapeutics. In this study, we tried to answer the questions whether TDP1 removes TOPO1 alone or with the help of other repair factors, and whether TDP1 can remove other proteins that are also cross- linked to the DNA backbone. To answer these questions, we created TDP1 deficient zebrafish models using CRISPR/Cas methods. In one cell stage embryos, we introduced a gRNA-Cas9 complex that specifically cleaves the DNA sequence for TDP1 protein which results in introduction of premature STOP codon in the beginning of the coding sequence. In order to generate catalytically inactive TDP1 protein, we have introduced C23572G i A23573C (H501A) mutations using CRISPR/Cas9 system and homologous recombination which incorporated repair template carrying target mutation in the zebrafish genome. In TDP1 deficient embryos, we have measured the presence and repair of TOPO1-DNA crosslinks after isolation with guanidine chloride (6 M) followed by detection TOPO1-DNA crosslink with TOPO1-DNA crosslink specific antibody and slot blot method. Using the CRISPR/Cas9 system and RNA silencing, we are performing corresponding measurements on human cells. We have shown that an increase in the concentration of camptothecin causes the accumulation of TOPO1- DNA complex. The phosphodiesterase activity of TDP1 protein is crucial for the repair of the TOPO1–DNA complex induced by the camptothecin, how this repair takes place and which factors are crucial will be shown by ongoing research.

TDP1 ; DNA repair ; SPRTN ; DPC repair

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