Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica
Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica
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DOI：
10.1186/1471-2199-9-35
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发表时间：
2008-04-10
影响因子：
--
通讯作者：
Lopez-Camarillo, Cesar
中科院分区：
文献类型：
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作者：
Lopez-Casamichana, Mavil;Orozco, Esther;Lopez-Camarillo, Cesar
背景：在真核和原核细胞中，同源重组是产生遗传多样性的精确机制，也用于修复DNA双链断裂。参与重组 DNA 修复的 RAD52 上位组基因，包括 mre11、rad50、nsb1/xrs2、rad51、rad51c/rad57、rad51b/rad55、rad51d、xrcc2、xrcc3、rad52、rad54、rad54b/rdh54 和 rad59 基因，已在人类和酵母细胞中进行了研究。值得注意的是，RAD51 重组酶催化断裂 DNA 与其未受损同源链之间的链转移，以修复受损区域。在原生动物寄生虫中，同源重组产生的抗原变异和基因组重排是毒力变异和耐药性的原因。然而，在溶组织内阿米巴中，负责人类阿米巴病的原生动物寄生虫、DNA修复和同源重组机制仍然未知。 结果：在本文中，我们利用UV-C（150 J/m(2)）照射的滋养体，开始研究原始真核生物溶组织内阿米巴中的同源重组DNA修复机制。通过 TUNEL 和彗星测定以及 EhH2AX 组蛋白磷酸化状态的评估，证实了受辐射细胞中的 DNA 双链断裂。在溶组织内阿米巴基因组中，我们鉴定了与酵母同源的基因和人类RAD52上位组基因，这些基因参与通过同源重组修复DNA双链断裂。有趣的是，溶组织内阿米巴RAD52上位性组相关基因在UV-C处理前后存在差异表达。接下来，我们重点关注假定的重组酶 EhRAD51 的表征，该酶保留了 RECA/RAD51 蛋白的典型结构。特异性抗体免疫检测核区和细胞质区室中的 EhRAD51 蛋白。此外，DNA损伤后，EhRAD51在溶组织内阿米巴滋养体中被定位为典型的核灶样结构。纯化的重组EhRAD51在体外表现出DNA结合和配对活性以及同源链之间的交换反应。结论：溶组织内阿米巴基因组中含有大部分RAD52上位组相关基因，这些基因在UV-C照射诱导DNA双链断裂时出现差异表达。为了响应 DNA 损伤，EhRAD51 蛋白过度表达并重新定位于核灶样结构中。功能分析证实 EhRAD51 是一种真正的重组酶。这些数据首次揭示了溶组织内阿米巴 RAD52 上位群基因和 EhRAD51 蛋白功能在这种古老真核寄生虫 DNA 损伤反应中的潜在作用。
Background: In eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. RAD52 epistasis group genes involved in recombinational DNA repair, including mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 and rad59 genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in Entamoeba histolytica the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown.Results: In this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote E. histolytica using UV-C ( 150 J/m(2)) irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In E. histolytica genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the E. histolytica RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear foci-like structures in E. histolytica trophozoites. Purified recombinant EhRAD51 exhibited DNA binding and pairing activities and exchanging reactions between homologous strands in vitro.Conclusion: E. histolytica genome contains most of the RAD52 epistasis group related genes, which were differentially expressed when DNA double strand-breaks were induced by UV-C irradiation. In response to DNA damage, EhRAD51 protein is overexpressed and relocalized in nuclear foci-like structures. Functional assays confirmed that EhRAD51 is a bonafide recombinase. These data provided the first insights about the potential roles of the E. histolytica RAD52 epistasis group genes and EhRAD51 protein function in DNA damage response of this ancient eukaryotic parasite.