Identification of <i>Plasmodium falciparum</i> DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function

The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium.真核生物减数分裂重组蛋白11（Meiotic Recombination protein 11, Mre11）在DNA损伤反应（DNA damage response, DDR）中发挥关键作用。具体而言，Mre11可感知并传导DNA双链断裂（DNA double strand breaks, DSB）信号，并通过隶属于同源重组（homologous recombination, HR）或非同源末端连接（non-homologous end joining, NHEJ）修复通路的效应蛋白促进断裂修复。在引发人类疟疾的恶性疟原虫（Plasmodium falciparum）中，已鉴定出HR和替代非同源末端连接（alternative-NHEJ）修复通路，但对于该生物体DNA损伤反应中的上游调控因子所知甚少。本研究在恶性疟原虫中鉴定出Mre11的推定同源蛋白（PfalMre11），其与人类Mre11的序列相似性为22%。同源建模结果显示，预测得到的PfalMre11核酸酶结构域与酿酒酵母（Saccharomyces cerevisiae）Mre11（ScMre11）的核酸酶结构域具有显著的结构相似性。功能互补实验表明，将PfalMre11的核酸酶结构域与酿酒酵母Mre11的C端结构域联合使用，可功能性互补Mre11缺陷型酵母菌株，从而证实了PfalMre11核酸酶活性的功能保守性。而PfalMre11核酸酶结构域内的氨基酸替换（D398N）则几乎完全废除了该功能互补活性。PfalMre11在恶性疟原虫有丝分裂活跃的滋养体和裂殖体阶段表达丰富，并可响应DNA损伤而上调，提示其在DNA损伤反应中发挥功能。PfalMre11可与PfalRad50发生物理相互作用。此外，酵母双杂交实验显示，PfalMre11可与酿酒酵母中参与DNA损伤反应信号传导与修复的研究较为透彻的Mre11-Rad50-Xrs2复合体的两个组分ScRad50和ScXrs2相互作用，进一步支持了PfalMre11在DNA损伤反应中的作用。综上，这些研究结果证实PfalMre11是疟原虫中DNA损伤反应的一个进化保守组分。