DNA Topoisomerase 3α Is Involved in Homologous Recombination Repair and Replication Stress Response in Trypanosoma cruzi

DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3 alpha is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3 alpha knockout parasite (TcTopo3 alpha KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3 alpha knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3 alpha KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3 alpha KO, and a higher number of dormant cells was observed. Additionally, TcTopo3 alpha KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3 alpha in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3 alpha KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3 alpha KO parasites showed a population with sub-G1 DNA content and strong gamma H2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3 alpha leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3 alpha KO cells owing to MMS treatment. Thus, topoisomerase 3 alpha is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.

Automated summary

DNA topoisomerases, in particular topoisomerase 3 alpha, play crucial roles in genomic maintenance, cell growth, and DNA damage response in Trypanosoma cruzi. Knockout of topoisomerase 3 alpha affects parasite growth, invasion rates, and ability to repair DNA damage induced by genotoxic agents. These findings underscore the importance of topoisomerase 3 alpha in homologous recombination repair and replication stress in T. cruzi.