Estudo dos mecanismos de sinalização e reparo do dano de DNA durante o estresse replicativo em Trypanosoma cruzi

Abstract

Unrepaired DNA damage, limited concentration of free dNTPs, or topological stress caused by DNA transcription may slow or stall the replication fork progression, which is known as replicative stress. DNA topoisomerase 3α dissolutes the Holliday junctions during homologous recombination and DNA topoisomerase 3β suppresses the accumulation of R-loops formed during transcription. Therefore, these enzymes can prevent replicative stress. Once replication forks have stopped their movement, eukaryotic cells rely on ATM and ATR kinases to coordinate cell cycle transitions, recruit repair proteins, and regulate apoptosis. Both topoisomerase 3α and 3β genes as well as ATM and ATR kinases are present in the trypanosomatid genome, but the functions of these proteins are unclear in Trypanosoma cruzi, the parasite that causes Chagas disease. Thus, the present work aimed to evaluate the role of these enzymes during the replicative stress in T. cruzi. To analyze the involvement of topoisomerase 3α in the homologous recombination repair, wild type parasite (WT) and knockout parasites (Topo3α KO and Topo3β KO) were exposed to gamma radiation. Only Topo3α KO cells were unable to resume growth after treatment, indicating the importance of this enzyme during this repair pathway. These cells were also treated with drugs that halt the replication fork. Topo3α KO parasites were more sensitive to treatment with cisplatin, hydroxyurea and methyl methane sulfonate (MMS) than WT and Topo3β KO parasites. However, it was observed a variation in the sensitivity of Topo3α KO cells during the treatments tested, which indicates the involvement of more than one repair pathway in topoisomerase 3α-mediated replicative stress resolution. A similar difference was also observed in the Rad51 deficient cells, the recombinase that acts on homologous recombination, which corroborates the participation of topoisomerase 3α in this repair pathway. To study the function of ATM and ATR kinases, wild type epimastigotes of CL Brener strain were transfected with a deletion cassette, which allows the generation of single knockout parasites for the target genes. After transfection, parasites with reduced ATM levels were correctly selected. The study of these cells will allow a better understanding of the mechanisms involved in the DNA double strand break response in T. cruzi.