DataSheet_1_Specific Human ATR and ATM Inhibitors Modulate Single Strand DNA Formation in Leishmania major Exposed to Oxidative Agent.zip

Leishmania parasites are the causative agents of a group of neglected tropical diseases known as leishmaniasis. The molecular mechanisms employed by these parasites to adapt to the adverse conditions found in their hosts are not yet completely understood. DNA repair pathways can be used by Leishmania to enable survival in the interior of macrophages, where the parasite is constantly exposed to oxygen reactive species. In higher eukaryotes, DNA repair pathways are coordinated by the central protein kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). The enzyme Exonuclease-1 (EXO1) plays important roles in DNA replication, repair, and recombination, and it can be regulated by ATM- and ATR-mediated signaling pathways. In this study, the DNA damage response pathways in promastigote forms of L. major were investigated using bioinformatics tools, exposure of lineages to oxidizing agents and radiation damage, treatment of cells with ATM and ATR inhibitors, and flow cytometry analysis. We demonstrated high structural and important residue conservation for the catalytic activity of the putative LmjEXO1. The overexpression of putative LmjEXO1 made L. major cells more susceptible to genotoxic damage, most likely due to the nuclease activity of this enzyme and the occurrence of hyper-resection of DNA strands. These cells could be rescued by the addition of caffeine or a selective ATM inhibitor. In contrast, ATR-specific inhibition made the control cells more susceptible to oxidative damage in an LmjEXO1 overexpression-like manner. We demonstrated that ATR-specific inhibition results in the formation of extended single-stranded DNA, most likely due to EXO1 nucleasic activity. Antagonistically, ATM inhibition prevented single-strand DNA formation, which could explain the survival phenotype of lineages overexpressing LmjEXO1. These results suggest that an ATM homolog in Leishmania could act to promote end resection by putative LmjEXO1, and an ATR homologue could prevent hyper-resection, ensuring adequate repair of the parasite DNA.

利什曼原虫是导致一组被称为利什曼病的被忽视的热带疾病的病原体。这些寄生虫适应宿主体内恶劣环境所采用的分子机制尚未完全明了。利什曼原虫可以利用DNA修复途径，在巨噬细胞内部生存，该处寄生虫持续暴露于活性氧物种中。在高等真核生物中，DNA修复途径由中央蛋白激酶ataxia telangiectasia mutated (ATM) 和 ataxia telangiectasia and Rad3 related (ATR) 协调。Exonuclease-1 (EXO1) 酶在DNA复制、修复和重组中扮演着重要角色，其活性可以受到ATM-和ATR介导的信号通路的调控。在本研究中，利用生物信息学工具、暴露细胞系于氧化剂和辐射损伤、使用ATM和ATR抑制剂处理细胞以及流式细胞术分析，对L. major的锥虫形态的DNA损伤反应途径进行了研究。我们证明了拟LmjEXO1的催化活性具有高度的结构和重要的残基保守性。拟LmjEXO1的过表达使得利什曼原虫细胞对致基因毒性损伤更为敏感，这很可能归因于该酶的核酸酶活性和DNA链过度重组的发生。这些细胞可以通过添加咖啡因或选择性ATM抑制剂得到挽救。相反，ATR特异性抑制使对照组细胞以拟LmjEXO1过表达类似的方式对氧化损伤更为敏感。我们证明了ATR特异性抑制会导致长链单链DNA的形成，这很可能归因于EXO1的核酸酶活性。相反，ATM抑制阻止了单链DNA的形成，这可以解释过表达LmjEXO1的细胞系的存活表型。这些结果表明，利什曼原虫中的ATM同源物可能通过拟LmjEXO1促进末端重组，而ATR同源物可以防止过度重组，确保寄生虫DNA得到充分的修复。