An intramolecular G-quadruplex structure is required for binding of telomeric repeat-containing RNA to the telomeric protein TRF2

Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization.     

Chemistry in human telomere biology: structure, function and targeting of telomere DNA/RNA

Telomeres are present at the ends of all eukaryotic chromosomes. Human telomeres play an important role in critical processes underlying genome stability, cancer, and aging, and their importance was recognized via the award of the 2009 Nobel Prize in Physiology or Medicine. Chemistry has made vast and almost unparalleled contributions to telomere biology. This critical review highlights the contributions of chemistry in human telomeres and summarizes the significant development of human telomere biology. First, I provide an overview of the advances in understanding of the structures and functions of human telomeres. Second, I focus on the current efforts on developing various chemical approaches to targeting human telomeres and telomerase for the treatment of cancer. Third, studies on a newly discovered telomeric repeat-containing RNA are discussed in detail. Last, future challenges in the field are outlined, including perspectives of both chemistry and biology (412 references).     

Decreased expression of DNA repair proteins Ku70 and Mre11 is associated with aging and may contribute to the cellular senescence

The gradual loss of telomeric DNA can contribute to replicative senescence and thus, having longer telomeric DNA is generally considered to provide a longer lifespan. Maintenance and stabilization of telomeric DNA is assisted by binding of multiple DNA-binding proteins, including those involved in double strand break (DSB) repair. We reasoned that declining DSB repair capacity and increased telomere shortening in aged individuals may be associated with decreased expression of DSB repair proteins capable of telomere binding. Our data presented here show that among the DSB repair proteins tested, only the expression of Ku70 and Mre11 showed statistically significant age-dependent changes in human lymphocytes. Furthermore, we found that expressions of Ku70 and Mre11 are statistically correlated, which indicate that the function of Ku70 and Mre11 may be related. All the other DSB repair proteins tested, Sir2, TRF1 and Ku80, did not show any significant differences upon aging. In line with these data, people who live in the regional community (longevity group), which was found to have statistically longer average life span than the rest area, shows higher level of Ku70 expression than those living in the neighboring control community. Taken together, our data show, for the first time, that Ku70 and Mre11 may represent new biomarkers for aging and further suggest that maintenance of higher expression of Ku70 and Mre11 may be responsible for keeping longer life span observed in the longevity group.     

Recombination proteins and telomere stability in plants

Repair of DNA damage is essential for the maintenance of the integrity and transmission of the genome in development and reproduction. Telomeres are nucleoprotein structures which protect the ends of (linear) eukaryotic chromosomes. Telomere dysfunction results in loss of this protection and the telomeres being recognised as DNA damage by the cellular DNA Damage Repair and Response (DDR) machinery, leading to senescence or cell death. Telomeric homeostasis is thus tightly controlled and many specific and non-specific proteins are involved in its regulation. Among these, DNA damage and Repair proteins contribute both to the recognition of telomere dysfunction and more surprisingly, are directly implicated in telomere homeostasis itself. Plants offer a great opportunity to study these mechanisms due to the fact that many key DNA repair and recombination proteins are non-essential in plants, in contrast to vertebrates. In the following text, after a brief summary of the current state of knowledge on telomere-specific proteins in plants, we review the DDR processes and the related proteins implicated in plant telomere stability. We focus specifically on telomere signalling and on recombination events induced by unprotected telomeres, at the origin of genome rearrangements and instability when telomere function is affected.     

Human telomeric DNA forms parallel-stranded intramolecular G-quadruplex in K+ solution under molecular crowding condition

The G-rich strand of human telomeric DNA can fold into a four-stranded structure called G-quadruplex and inhibit telomerase activity that is expressed in 85-90% tumor cells. For this reason, telomere quadruplex is emerging as a potential therapeutic target for cancer. Information on the structure of the quadruplex in the physiological environment is important for structure-based drug design targeting the quadruplex. Recent studies have raised significant controversy regarding the exact structure of the quadruplex formed by human telomeric DNA in a physiological relevant environment. Studies on the crystal prepared in K+ solution revealed a distinct propeller-shaped parallel-stranded conformation. However, many later works failed to confirm such structure in physiological K+ solution but rather led to the identification of a different hybrid-type mixed parallel/antiparallel quadruplex. Here we demonstrate that human telomere DNA adopts a parallel-stranded conformation in physiological K+ solution under molecular crowding conditions created by PEG. At the concentration of 40% (w/v), PEG induced complete structural conversion to a parallel-stranded G-quadruplex. We also show that the quadruplex formed under such a condition has unusual stability and significant negative impact on telomerase processivity. Since the environment inside cells is molecularly crowded, our results obtained under the cell mimicking condition suggest that the parallel-stranded quadruplex may be the more favored structure under physiological conditions, and drug design targeting the human telomeric quadruplex should take this into consideration.     

Insight into How Telomeric G‐Quadruplexes Enhance the Peroxidase Activity of Cellular Hemin

The toxic oxidative damage of G‐quadruplexes (G4), linked to neurodegenerative diseases, may arise from their ability to bind and oxidatively activate cellular hemin. However, there have been no precise studies on how telomeric G4 enhances the low intrinsic peroxidase activity of hemin. Herein, a label‐free and nanopore‐based strategy was developed to explore the enhancement mechanism of peroxidase activity of hemin induced by telomeric G4 (d(TTAGGG)_n). The nanopore‐based strategy demonstrated that there were simultaneously two different binding modes of telomere G4 to hemin. At the single‐molecule level, it was found that the hybrid structural telomeric G4 directly binds to hemin (the affinity constant (K_a)≈10⁶ m ^?1) to form a tight complex, and some of them underwent a topological change to a parallel structure with an enhancement of K_a to approximately 10⁷ m ^?1. Through detailed analysis of the topology and peroxidase activity and molecular modeling investigations, the parallel telomere G4/hemin DNAzyme structure was proven to be preferable for high peroxidase activity. Upon strong π–π stacking, the parallel structural telomere G4 supplied a key axial ligand to the hemin iron, which accelerated the intermediate compound formation with H₂O₂ in the catalytic cycle. Our studies developed a label‐free and single‐molecule strategy to fundamentally understand the catalytic activity and mechanism of telomeric DNAzyme, which provides some support for utilizing the toxic, oxidative‐damage property in cellular oxidative disease and anticancer therapeutics.     

Signal modelization for improved precision of assessment of minimum and mean telomere lengths

Telomere length is an important measure of cell and tissue regenerative capacities. The mean telomere length is classically used as global indicator of a tissue telomere length. In skeletal muscle, which is made of postmitotic myonuclei and satellite cells (muscle stem cells), minimum telomere length is also used to assess the telomere length of satellite cells and newly incorporated myonuclei. At present, the estimation of the method reproducibility during the assessment of mean and minimum telomere length using Southern blot analysis has never been documented. The aim of this report is to describe a signal modelization for improved precision of assessment of minimum and mean telomere lengths and to document the method reproducibility. Telomeres are assessed using a Southern technique where the gel is directly hybridized with the specific probe without the membrane-transferring step in order to prevent telomeric low signal loss. We found that the improved signal analysis for determination of telomere length is associated with coefficients of variation ranging from 1.37 to 4.29% for the mean telomeric restriction fragment (TRF) length and from 2.04 to 4.95% for the minimum TRF length. Improved method reproducibility would allow saving time and biological material as duplicate and triplicate measurement of the same sample is no longer required.     

(3 + 1) Assembly of three human telomeric repeats into an asymmetric dimeric G-quadruplex

We present an NMR study on the structure of a DNA fragment of the human telomere containing three guanine-tracts, d(GGGTTAGGGTTAGGGT). This sequence forms in Na(+) solution a unique asymmetric dimeric quadruplex, in which the G-tetrad core involves all three G-tracts of one strand and only the last 3'-end G-tract of the other strand. We show that a three-repeat human telomeric sequence can also associate with a single-repeat human telomeric sequence into a structure with the same topology that we name (3 + 1) quadruplex assembly. In this G-quadruplex assembly, there are one syn.syn.syn.anti and two anti.anti.anti.syn G-tetrads, two edgewise loops, three G-tracts oriented in one direction and the fourth oriented in the opposite direction. We discuss the possible implications of the new folding topology for understanding the structure of telomeric DNA, including t-loop formation, and for targeting G-quadruplexes in the telomeres.     

Structural,functional, and evolutionary analysis of late embryogenesis abundant proteins (LEA) in Triticum aestivum: A detailed molecular level biochemistry using in silico approach

LEA (Late Embryogenesis Abundant) proteins are abundant in plants and play a crucial role in abiotic stress tolerance. In our work, we primarily focused on the variations in physiochemical properties, conserved domains, secondary structure, gene ontology and evolutionary relationships among 40 LEA proteins of Triticum aestivum (common wheat). Wheat LEA protein belongs to first 6 classes out of the 13 classes present in LEApdB, the comprehensive database for LEA proteins. Proteins belonging to each LEApdB class have structures and functions distinguished from other classes. The study found three different conserved LEA domains in Triticum aestivum. One important domain was dehydrin, present in wheat proteins of classes 1, 2 and 4, though varied in sequence level, have similar biological processes. The study also found sequence level and phylogenetic similarity between dehydrin domains of class 1 and 4, but distinct from that of LEApdB class 2. This study also demonstrated functional diversity in two class 6 proteins occurred due to many destabilizing mutations in the LEA4 domain that caused alteration of ligand binding and conformational shift from 3₁₀-helix → turn within the domain. The LEA4 domains of these proteins also showed functional similarity and evolutionary relatedness with three other proteins of genus Aegilops, denoting that these proteins in Triticum aestivum were derived from its ancestor Aegilops. The study also assigned LEApdB class 4 to an unclassified LEA protein ‘WZY2-1’ based on amino acid composition, conserved domain, motif architecture and phylogenetic relatedness with class 4 proteins. Our study has revealed a detailed analysis of LEA proteins in Triticum aestivum and can serve as a pillar for further investigations and comparative analysis of wheat LEA proteins with other cereal or plant types.     

Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis

To increase the number of proteins detectable by two-dimensional electrophoresis (2-DE) in plants, we present a new procedure for extracting total proteins from plant tissue. This method avoids any loss of proteins in the course of sample preparation and results in two different fractions, one comprising mainly the cytoplasmatic proteins, the other one containing predominantly structure bond proteins. 2-DE patterns obtained from these two fractions show that the total number of different protein spots detected exceeds the degree of resolution commonly reported for plant proteins threefold.     

Multidomain protein solves the folding problem by multifunnel combined landscape: theoretical investigation of a y-family DNA polymerase

Approximately three-fourths of eukaryotic proteins are composed of multiple independently folded domains. However, much of our understanding is based on single domain proteins or isolated domains whose studies directly lead to well-known energy landscape theory in which proteins fold by navigating through a funneled energy landscape toward native structure ensembles. The degrees of freedom for proteins with multiple domains are many orders of magnitude larger than that for single domain proteins. Now, the question arises: How do the multidomain proteins solve the "protein folding problem"? Here, we specifically address this issue by exploring the structure folding relationship of Sulfolobus solfataricus DNA polymerase IV (DPO4), a prototype Y-family DNA polymerase which contains a polymerase core consisting of a palm (P domain), a finger (F domain), and a thumb domain (T domain) in addition to a little finger domain (LF domain). The theoretical results are in good agreement with the experimental data and lead to several theoretical predictions. Finally, we propose that for rapid folding into well-defined conformations which carry out the biological functions, four-domain DPO4 employs a divide-and-conquer strategy, that is, combining multiple individual folding funnels into a single funnel (domains fold independently and then coalesce). In this way, the degrees of freedom for multidomain proteins are polynomial rather than exponential, and the conformational search process can be reduced effectively from a large to a smaller time scale.     

Telomere and Telomerase as Targets for Cancer Therapy

Telomere maintenance and telomerase reactivation is essential for the transformation of most human cancer cells. Telomere shortening to the threshold length, mutations of the telomere-associated proteins, and/or telomerase RNA lead to telomeric dysfunction and therefore genomic instability. Telomerase up-regulation in 85% of human cancer cells has become a hallmark of cancers, hence a promising target for anticancer therapy. In this review, we discuss the mechanism of cancer due to telomere dysfunction and the resulting biological effects, the control of telomerase activity, and the new developments in cancer therapies targeting telomere and telomerase.     

Conformational fluctuations of a protein-DNA complex and the structure and ordering of water around it

Protein-DNA binding is an important process responsible for the regulation of genetic activities in living organisms. The most crucial issue in this problem is how the protein recognizes the DNA and identifies its target base sequences. Water molecules present around the protein and DNA are also expected to play an important role in mediating the recognition process and controlling the structure of the complex. We have performed atomistic molecular dynamics simulations of an aqueous solution of the protein-DNA complex formed between the DNA binding domain of human TRF1 protein and a telomeric DNA. The conformational fluctuations of the protein and DNA and the microscopic structure and ordering of water around them in the complex have been explored. In agreement with experimental studies, the calculations reveal conformational immobilization of the terminal segments of the protein on complexation. Importantly, it is discovered that both structural adaptations of the protein and DNA, and the subsequent correlation between them to bind, contribute to the net entropy loss associated with the complex formation. Further, it is found that water molecules around the DNA are more structured with significantly higher density and ordering than that around the protein in the complex.     

Comparative binding of antitumor drugs to DNA containing the telomere repeat sequence

Telomeres are the ends of the linear chromosomes of eukaryotes and consist of tandem GT-rich repeats in telomere sequence i.e. 500-3000 repeats of 5'-TTAGGG-3' in human somatic cells, which are shortened gradually with age. The G-rich overhang of telomere sequence can adopt different intramolecular fold-backs and tetra-stranded DNA structures, in vitro, which inhibit telomerase activity. In this report, DNA binding agents to telomere sequence were studied novel therapeutic possibility to destabilize telomeric DNA sequences. Oligonucleotides containing the guanine repeats in human telomere sequence were synthesized and used for screening potential antitumor drugs. Telomeric DNA sequence was characterized using spectral measurements and CD spectroscopy. CD spectrum indicated that the double-stranded telomeric DNA is in a right-handed conformation. Polyacrylamide gel electrophoresis was performed for binding behaviors of antitumor compounds with telomeric DNA sequence. Drugs interacted with DNA sequence caused changes in the electrophoretic mobility and band intensity of the gels. Depending on the binding mode of the anticancer drugs, telomeric DNA sequence was differently recognized and the efficiency of cleavage of DNA varies in the bleomycin-treated samples under different conditions. DNA cleavage occurred at about 1% by the increments of 1 micromM bleomycin-Fe(III). These results imply that the stability of human telomere sequence is important in conjunction with the cancer treatment and aging process.     

Proteomic Analysis of Differentially Expressed Proteins of Arabidopsis thaliana Response to Specialist Herbivore Plutella xylostella

The changes of the proteome in Arabidopsis thaliana leaves were examined by specialist Plutella xylostella.Analysis of about 1100 protein spots on each 2DE gel revealed 38 differentially expressed protein spots in abun-dance of which 34 proteins were identified by MALDI-TOF/TOF MS.Among the insect feeding responsive proteins,a few proteins involved in carbon metabolism were identified including proteins associated with the Calvin cycle in the chloroplast and TCA cycle in the mitochondria,indicating carbon metabolism related proteins may play crucial roles in induced defense response in plants under insect infestation.The analysis elucidates the subcellular location of proteins demonstrates that about 50% of proteins are in the chloroplast,which shows the chloroplast has a key role in the insect feeding response for plant.Gene expression analysis of 10 different proteins by quantitative real-time PCR shows that four proteins of the mRNA level were correlated well with the protein level.This study further dissected the nature of insect infestation as a stress signal and some novel insect feeding responsive proteins identified may play an important role in induced defence machanism for plant.