A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis
A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis
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DOI：
10.1016/j.bbagen.2020.129607
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发表时间：
2020-07-01
影响因子：
3
通讯作者：
Cano, Maria Isabel Nogueira
中科院分区：
文献类型：
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作者：
Fernandes, Carlos A. H.;Morea, Edna Gicela O.;Cano, Maria Isabel Nogueira
背景：端粒是维持基因组动态平衡的重要染色体末端结构。它们由端粒酶和相关蛋白的作用来补充，如含有端粒末端结合蛋白的OB(oligonucleotide/oligosaccharide-binding)-fold，它在端粒的维持和保护中起着至关重要的作用。TEBP的性质在高等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。以往的计算机检索表明，在包括利什曼原虫在内的锥虫中没有经典的TEPB的同源物。然而，复制蛋白A亚单位1(RPA-1)是一种包含DNA结合蛋白的OB折叠蛋白，它与锥虫端粒共定位，并显示出对端粒富含G链的高度偏好。方法和结果：我们预测利什曼原虫中缺少包含TEBP的OB折叠结构同源蛋白。基因组的结构比较。通过分子对接，我们证明了LaRPA-1的单链DNA结合模式与高等真核生物POT1和RPA-1的晶体结构单链DNA结合模式具有相同的特征。通过荧光光谱、蛋白质-DNA相互作用分析和FRET，我们分别证明了LaRPA-1与经典的TEBP具有相同的端粒功能，因为它可以结合至少一个端粒重复序列，保护富含G的端粒DNA不被3‘-5’外切酶I消化，并展开端粒G-四链。结论：我们的结果表明RPA-1在锥虫中以TEBP的形式存在，在此背景下，我们提出了两种可能的锥虫RPA-1的进化图景，可以反映所有真核生物中含有OB-折叠的TEBP的进化。
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.