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

Abstract

DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α 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 3α knockout parasite (TcTopo3α 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 TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α 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 TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α 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, TcTopo3α 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. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α 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 TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α 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.

Searching for G-Quadruplex-Binding Proteins in Plants: New Insight into Possible G-Quadruplex Regulation

“…These proteins can be divided into three groups: proteins able to bind/interact with DNA or RNA; proteins linked with ribosome biogenesis and maintenance; other proteins. The most interesting proteins from the first group (proteins able to interact with DNA or RNA) were the following: DNA topoisomerase 3α (involved in homologous recombination and torsion stretch relaxation [ 35 ]) and the plant-unique protein KAKU4 (responsible for nuclear morphology in plants [ 36 ]). In addition, our data also indicate the G4-interacting potential for the following: TF II D-subunit 15b (transcription initiation factor), RNA-binding family protein (RRM/RBD/RNP motifs) responsible for RNA splicing, the serine/arginine-rich splicing factor RSZ21 (mRNA processing [ 37 ]), the mediator of RNA pol II-transcription subunit 36a (mediator of RNA polymerase II), protein decapping 3α (translation repressor), and proteins belonging to the Argonaute family (AGO2 and AGO3, which contain the NIQI motif).…”

Temporal and Wash-Out Studies Identify Medicines for Malaria Venture Pathogen Box Compounds with Fast-Acting Activity against Both Trypanosoma cruzi and Trypanosoma brucei

“…Whilst pyrazoloquinazolines do not have reported activity against trypanosomes, compounds from this class have demonstrated inhibition of human topoisomerase I [ 60 ]. Topoisomerase has been identified as a target in trypanosomes and has recently been implicated in response to replication stress in T. cruzi [ 61 ].…”

Trypanosoma cruzi Genomic Variability: Array Comparative Genomic Hybridization Analysis of Clone and Parental Strain

“…They showed that amastigote and epimastigote dormancy is strain-dependent in T. cruzi and it is directly correlated with mRNA TcRAD51 levels, a recombination pivot, responsible for strand invasion and search for homology between broken strand and the template (Baumann and West, 1998;Gomes Passos Silva et al, 2018;Resende et al, 2020). Costa-Silva et al (2021) demonstrated that DNA topoisomerase 3a (TcTopo3a) is important for homologous recombination repair and replication stress in T. cruzi. TcTopo3a gene knockout inhibited the amastigote proliferation and a high number of dormant cells was identified.…”