Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
Division of Molecular Parasitology and Immunology, CSIR-Central Drug Research Institute, Lucknow, India.
DNA Repair (Amst). 2021 May;101:103098. doi: 10.1016/j.dnarep.2021.103098. Epub 2021 Mar 14.
The malaria parasite has a single mitochondrion which carries multiple tandem repeats of its 6 kb genome encoding three proteins of the electron transport chain. There is little information about DNA repair mechanisms for mitochondrial genome maintenance in Plasmodium spp. Of the two AP-endonucleases of the BER pathway encoded in the parasite nuclear genome, the EndoIV homolog PfApn1 has been identified as a mitochondrial protein with restricted functions. We explored the targeting and biochemical properties of the ExoIII homolog PfApe1. PfApe1 localized in the mitochondrion and exhibited AP-site cleavage, 3'-5' exonuclease, 3'-phosphatase, nucleotide incision repair (NIR) and RNA cleavage activities indicating a wider functional role than PfApn1. The parasite enzyme differed from human APE1 in possessing a large, disordered N-terminal extension. Molecular modelling revealed conservation of structural domains but variations in DNA-interacting residues and an insertion in the α-8 loop suggested differences with APE1. Unlike APE1, where AP-site cleavage and NIR activities could be mutually exclusive based on pH and Mg ion concentration, PfApe1 was optimally active under similar conditions suggesting that it can function both as an AP-endonuclease in BER and directly cleave damaged bases in NIR under similar physiological conditions. To investigate the role of Ape1 in malaria life cycle, we disrupted the gene by double-cross-over homologous recombination. Ape1 knockout (KO) P. berghei parasites showed normal development of blood and mosquito stages. However, inoculation of mice with Ape1 KO salivary gland sporozoites revealed a reduced capacity to initiate blood stage infection. Ape1 KO parasites underwent normal liver stage development until merozoites egressed from hepatocytes. Our results indicated that the delay in pre-patent period was due to the inability of Ape1 KO merosomes to infect erythrocytes efficiently.
疟原虫只有一个线粒体，其中携带其 6kb 基因组的多个串联重复序列，这些序列编码电子传递链的三种蛋白质。关于疟原虫属维持线粒体基因组的 DNA 修复机制，信息很少。在寄生虫核基因组中编码的 BER 途径的两个 AP 内切酶中，已鉴定出 EndoIV 同源物 PfApn1 为具有受限功能的线粒体蛋白。我们探讨了 ExoIII 同源物 PfApe1 的靶向和生化特性。PfApe1 定位于线粒体中，表现出 AP 位点切割、3'-5'外切核酸酶、3'-磷酸酶、核苷酸切口修复 (NIR) 和 RNA 切割活性，表明其功能比 PfApn1 更广泛。寄生虫酶与人类 APE1 不同，它具有较大的、无序的 N 端延伸。分子建模揭示了结构域的保守性，但 DNA 相互作用残基的变化和α-8 环中的插入表明与 APE1 存在差异。与 APE1 不同，AP 位点切割和 NIR 活性可以根据 pH 值和 Mg 离子浓度相互排斥，PfApe1 在相似的条件下表现出最佳活性，表明它可以在 BER 中作为 AP 内切酶发挥作用，并在相似的生理条件下直接在 NIR 中切割受损碱基。为了研究 Ape1 在疟原虫生命周期中的作用，我们通过双交叉同源重组破坏了该基因。Ape1 敲除 (KO) P. berghei 寄生虫的血液和蚊子阶段发育正常。然而，用 Ape1 KO 唾液腺子孢子接种小鼠表明，其开始血液阶段感染的能力降低。Ape1 KO 寄生虫经历了正常的肝期发育，直到裂殖子从肝细胞中逸出。我们的结果表明，潜育期的延迟是由于 Ape1 KO 疟原虫体不能有效地感染红细胞。
