Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum
Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum
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DOI：
10.1128/mbio.00252-13
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发表时间：
2013-05-01
期刊：
影响因子：
6.4
通讯作者：
Kumar, Nirbhay
中科院分区：
文献类型：
--
作者：
Gopalakrishnan, Anusha M.;Kumar, Nirbhay
细菌RecA蛋白及其真核同源物Rad51在重组和DNA修复过程中的同源DNA链交换反应中发挥核心作用。以前，我们的实验室已经表明，PfRad51，恶性疟原虫Rad51同源物，表现出ATP酶活性，并促进DNA链交换在体外。在这项研究中，我们评估了PfRad51在假定的相互作用伴侣，特别是Rad54和复制蛋白A的恶性疟原虫同源物存在下的催化功能。PfRad54加速PfRad51介导的配对之间的单链DNA（ssDNA）和它的同源线性双链DNA（dsDNA）在0.5 mM CaCl2的存在下。我们还提出的证据表明，重组PfRPA1L蛋白的细菌同源单链结合蛋白（SSB）在启动同源配对和链交换活性的功能。更重要的是，PfRPA1L的功能以剂量依赖性方式受到PfRPA1S的负调控，PfRPA1S是恶性疟原虫中的另一种RPA同源物。最后，我们提出了在体内的证据，通过彗星试验甲基甲烷磺酸诱导的DNA损伤的疟疾寄生虫和伴随上调PfRad51，PfRad54，PfRPA1L，PfRPA1S在转录和蛋白质水平需要修复DNA损伤。本研究提供了新的见解，假定Rad51相互作用蛋白参与同源重组的作用，并强调在寄生虫的生长过程中的DNA损伤修复的生理作用。重要意义同源重组中起着重要作用的染色体重排，Rad51蛋白，辅助几个其他蛋白质，在重组和DNA修复过程中的DNA链交换反应中起着核心作用。本研究报道了恶性疟原虫Rad51在同源链交换和DNA修复中的作用的表征，并评估了PfRad54和PfRPA 1蛋白的功能贡献。这里提出的数据提供了深入了解这种寄生虫的DNA重组和DNA损伤修复机制的机制。这些研究结果在未来工作中的重要性将是研究Rad51依赖性机制是否参与恶性疟原虫抗原变异过程中的染色体重排。抗原变异的一个重要决定因素，即寄生虫快速改变其表面分子的非凡能力，与var基因有关，抗原变异对疫苗开发提出了重大挑战。
The bacterial RecA protein and its eukaryotic homologue Rad51 play a central role in the homologous DNA strand exchange reaction during recombination and DNA repair. Previously, our lab has shown that PfRad51, the Plasmodium falciparum homologue of Rad51, exhibited ATPase activity and promoted DNA strand exchange in vitro. In this study, we evaluated the catalytic functions of PfRad51 in the presence of putative interacting partners, especially P. falciparum homologues of Rad54 and replication protein A. PfRad54 accelerated PfRad51-mediated pairing between single-stranded DNA (ssDNA) and its homologous linear double-stranded DNA (dsDNA) in the presence of 0.5 mM CaCl2. We also present evidence that recombinant PfRPA1L protein serves the function of the bacterial homologue single-stranded binding protein (SSB) in initiating homologous pairing and strand exchange activity. More importantly, the function of PfRPA1L was negatively regulated in a dose-dependent manner by PfRPA1S, another RPA homologue in P. falciparum. Finally, we present in vivo evidence through comet assays for methyl methane sulfonate-induced DNA damage in malaria parasites and accompanying upregulation of PfRad51, PfRad54, PfRPA1L, and PfRPA1S at the level of transcript and protein needed to repair DNA damage. This study provides new insights into the role of putative Rad51-interacting proteins involved in homologous recombination and emphasizes the physiological role of DNA damage repair during the growth of parasites.IMPORTANCE Homologous recombination plays a major role in chromosomal rearrangement, and Rad51 protein, aided by several other proteins, plays a central role in DNA strand exchange reaction during recombination and DNA repair. This study reports on the characterization of the role of P. falciparum Rad51 in homologous strand exchange and DNA repair and evaluates the functional contribution of PfRad54 and PfRPA1 proteins. Data presented here provide mechanistic insights into DNA recombination and DNA damage repair mechanisms in this parasite. The importance of these research findings in future work will be to investigate if Rad51-dependent mechanisms are involved in chromosomal rearrangements during antigenic variation in P. falciparum. A prominent determinant of antigenic variation, the extraordinary ability of the parasite to rapidly change its surface molecules, is associated with var genes, and antigenic variation presents a major challenge to vaccine development.