A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis.,Biochimica et Biophysica Acta (BBA) - General Subjects

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A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis.
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 2.8 ) Pub Date : 2020-03-25 , DOI: 10.1016/j.bbagen.2020.129607
Carlos A H Fernandes ₁ , Edna Gicela O Morea ₂ , Gabriel A Dos Santos ₂ , Vitor L da Silva ₂ , Marina Roveri Vieira ₂ , Maria Alejandra Viviescas ₂ , Jean Chatain ₃ , Aurélie Vadel ₃ , Carole Saintomé ₄ , Marcos Roberto M Fontes ₅ , Maria Isabel Nogueira Cano ₂

Affiliation

BACKGROUND Telomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand. METHODS AND RESULTS We predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3'-5' Exonuclease I digestion, and unfold telomeric G-quadruplex. CONCLUSIONS Our results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes.

中文翻译：

多方法分析强调了 RPA-1 作为亚马逊利什曼原虫中端粒末端结合蛋白 (TEBP) 的相关性。

背景技术端粒是在维持基因组稳态中重要的染色体末端结构。它们通过端粒酶和相关蛋白的作用得到补充，例如包含在端粒维持和保护中起重要作用的端粒末端结合蛋白 (TEBP) 的 OB（寡核苷酸/寡糖结合）折叠。TEBPs 的性质在高等真核生物和一些原始真核生物中是众所周知的，但在锥虫中仍未确定。以前的计算机搜索表明，锥虫类中没有经典 TEPB 的同源物，包括 Leishmania sp。然而，复制蛋白 A 亚基 1 (RPA-1) 是一种含有 DNA 结合蛋白的 OB 折叠，被发现与锥虫端粒共定位，并显示出对富含 G 端粒的链的高度偏好。方法和结果我们预测在利什曼原虫中不存在含有 TEBP 的 OB 折叠结构同源物。使用结构比较的基因组。我们通过分子对接证明了 LaRPA-1 的 ssDNA 结合模式与高等真核生物 POT1 和 RPA-1 晶体结构 ssDNA 结合模式具有共同特征。使用荧光光谱、蛋白质-DNA 相互作用分析和 FRET，我们分别表明 LaRPA-1 与经典 TEBP 共享一些端粒功能，因为它可以结合至少一个端粒重复，保护端粒富含 G 的 DNA 免受 3'-5 ' 核酸外切酶 I 消化，并展开端粒 G-四链体。结论我们的研究结果表明，RPA-1 在锥虫中作为 TEBP 出现，在这种情况下，

更新日期：2020-03-26

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