Costa-Silva Héllida Marina, Resende Bruno Carvalho, Umaki Adriana Castilhos Souza, Prado Willian, da Silva Marcelo Santos, Virgílio Stela, Macedo Andrea Mara, Pena Sérgio Danilo Junho, Tahara Erich Birelli, Tosi Luiz Ricardo Orsini, Elias Maria Carolina, Andrade Luciana Oliveira, Reis-Cunha João Luís, Franco Glória Regina, Fragoso Stenio Perdigão, Machado Carlos Renato
Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Laboratório de Biologia Molecular e Sistêmica de Tripanossomatídeos, Instituto Carlos Chagas, Fundação Oswaldo Cruz (FIOCRUZ), Curitiba, Brazil.
Front Cell Dev Biol. 2021 May 13;9:633195w. doi: 10.3389/fcell.2021.633195. eCollection 2021.
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 , 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 , even though all the pathways in which this enzyme participates during the replication stress response remains elusive.
DNA拓扑异构酶是调节DNA拓扑结构的酶。其中，拓扑异构酶3α参与基因组维护，作用于DNA复制终止、姐妹染色单体分离以及重组中间体的溶解。为了评估该酶在恰加斯病病原体中的作用，构建了拓扑异构酶3α基因敲除寄生虫（TcTopo3α KO），并评估了寄生虫的生长情况及其对几种DNA损伤剂的反应。TcTopo3α基因敲除对无鞭毛体形式的生长没有影响，但观察到休眠率较高。与野生型谱系相比，TcTopo3α KO锥鞭毛体形式在LLC-MK2细胞中的侵袭率降低。无鞭毛体增殖在TcTopo3α KO中也受到损害，并且观察到休眠细胞数量更多。此外，TcTopo3α KO无鞭毛体在γ射线照射后无法恢复细胞生长，这表明拓扑异构酶3α参与同源重组。这些寄生虫对产生复制应激的药物也敏感，如顺铂（Cis）、羟基脲（HU）和甲磺酸甲酯（MMS）。在HU和顺铂处理后，TcTopo3α KO寄生虫显示细胞生长较慢，并且无法有效修复这些基因毒性剂诱导的DNA损伤。MMS处理后观察到的细胞生长表型与γ射线照射后相似，尽管MMS暴露后休眠细胞较少。MMS处理48小时后，TcTopo3α KO寄生虫显示出亚G1期DNA含量群体和强烈的γH2A信号。因此，由于缺乏TcTopo3α导致的DNA损伤细胞增殖可能导致细胞死亡。对MMS处理的寄生虫进行全基因组测序显示，与未处理的基因敲除寄生虫相比，多基因家族DFG-1和RHS的含量显著降低，并且22号染色体的亚端粒区域可能受到侵蚀。Southern印迹实验表明端粒缩短，这可能表明由于MMS处理，TcTopo3α KO细胞中存在基因组不稳定。因此，拓扑异构酶3α对于恰加斯病病原体中的同源重组修复和复制应激很重要，尽管该酶在复制应激反应中参与的所有途径仍然不清楚。
