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

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Costa-Silva, Hellida Marina ^[1] ; Resende, Bruno Carvalho ^[1] ; Umaki, Adriana Castilhos Souza ^[2] ; Prado, Willian ^[1] ; da Silva, Marcelo Santos ^[3] ; Virgilio, Stela ^[4] ; Macedo, Andrea Mara ^[1] ; Pena, Sergio Danilo Junho ^[1] ; Tahara, Erich Birelli ^[1] ; Tosi, Luiz Ricardo Orsini ^[4] ; Elias, Maria Carolina ^[3] ; Andrade, Luciana Oliveira ^[5] ; Reis-Cunha, Joao Luis ^[6] ; Franco, Gloria Regina ^[1] ; Fragoso, Stenio Perdigao ^[2] ; Machado, Carlos Renato ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Lab Genet Bioquim, Belo Horizonte, MG - Brazil

^[2] Fundacao Oswaldo Cruz FIOCRUZ, Inst Carlos Chagas, Lab Biol Mol & Sistem Tripanossomatideos, Curitiba, Parana - Brazil

^[3] Inst Butantan, Ctr Toxinas Resposta Imune & Sinalizacao Celular, Lab Ciclo Celular, Sao Paulo - Brazil

^[4] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Biol Celular & Mol & Bioagentes Patogen, Lab Biol Mol Leishmanias, Ribeirao Preto - Brazil

^[5] Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Morfol, Lab Biol Celular & Mol, Belo Horizonte, MG - Brazil

^[6] Univ Fed Minas Gerais, Escola Vet, Dept Med Vet Prevent, Belo Horizonte, MG - Brazil

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY; v. 9, MAY 13 2021.
• Citações Web of Science: 0

Resumo

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. (AU)

• Processo FAPESP: 14/24170-5 - Dinâmica da replicação do DNA em Trypanosoma cruzi: caracterização do licenciamento e da taxa de replicação
• Beneficiário: Marcelo Santos da Silva
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 18/14398-0 - Centro Reino-Unido-Brasil para o Estudo da Leishmaniose (JCPiL)
• Beneficiário: Angela Kaysel Cruz
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 13/07467-1 - CeTICS - Centro de Toxinas, Imuno-Resposta e Sinalização Celular
• Beneficiário: Hugo Aguirre Armelin
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 16/50050-2 - How do common and diverged features of the replicative stress response shape the biology of TriTryp parasites?
• Beneficiário: Maria Carolina Quartim Barbosa Elias Sabbaga
• Modalidade de apoio: Auxílio à Pesquisa - Temático