Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.

• MeSH
• Helminths genetics   MeSH
• G-Quadruplexes * MeSH
• Genome MeSH
• Nematoda * genetics   MeSH
• Humans MeSH
• Ligands MeSH
• Parasites genetics   MeSH
• Platyhelminths * genetics   MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ligands MeSH

We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its 'circular' nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).

• MeSH
• Nucleic Acid Conformation MeSH
• DNA, Circular chemistry   MeSH
• Models, Molecular MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Nucleotide Motifs MeSH
• Base Pairing MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Stereoisomerism MeSH
• Telomere chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Circular MeSH

The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

• Keywords
• Cdc13, G-hairpin, G-quadruplex, Saccharomyces cerevisiae, cell cycle, folding kinetics, telomerase, telomere,
• MeSH
• DNA-Binding Proteins metabolism   MeSH
• DNA metabolism   MeSH
• G-Quadruplexes MeSH
• Telomere Homeostasis physiology   MeSH
• DNA, Single-Stranded metabolism   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation MeSH
• Oligonucleotides genetics   MeSH
• Telomere-Binding Proteins metabolism   MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Telomerase genetics   MeSH
• Telomere metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cdc13 protein, S cerevisiae MeSH Browser
• DNA-Binding Proteins MeSH
• DNA MeSH
• DNA, Single-Stranded MeSH
• Oligonucleotides MeSH
• Telomere-Binding Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Telomerase MeSH

i-Motif (iM) is a four stranded DNA structure formed by cytosine-rich sequences, which are often present in functionally important parts of the genome such as promoters of genes and telomeres. Using electronic circular dichroism and UV absorption spectroscopies and electrophoretic methods, we examined the effect of four naturally occurring DNA base lesions on the folding and stability of the iM formed by the human telomere DNA sequence (C3TAA)3C3T. The results demonstrate that the TAA loop lesions, the apurinic site and 8-oxoadenine substituting for adenine, and the 5-hydroxymethyluracil substituting for thymine only marginally disturb the formation of iM. The presence of uracil, which is formed by enzymatic or spontaneous deamination of cytosine, shifts iM formation towards substantially more acidic pH values and simultaneously distinctly reduces iM stability. This effect depends on the position of the damage sites in the sequence. The results have enabled us to formulate additional rules for iM formation.

• MeSH
• Adenine analogs & derivatives   chemistry   MeSH
• Cytosine chemistry   MeSH
• DNA chemistry   MeSH
• Humans MeSH
• Pentoxyl analogs & derivatives   chemistry   MeSH
• DNA Damage MeSH
• Telomere chemistry   MeSH
• Uracil chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-hydroxymethyluracil MeSH Browser
• 8-hydroxyadenine MeSH Browser
• Adenine MeSH
• Cytosine MeSH
• DNA MeSH
• Pentoxyl MeSH
• Uracil MeSH

C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of living cells is not currently known. Herein, using state-of-the-art in-cell NMR spectroscopy, we evaluate the stabilities of i-motif structures in the complex cellular environment. We show that i-motifs formed from naturally occurring C-rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i-motif stabilities in vivo are generally distinct from those in vitro. Our results are the first to interlink the stability of DNA i-motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNA biosensors for intracellular applications.

• Keywords
• DNA, i-motifs, in-cell NMR spectroscopy, structural biology,
• MeSH
• Biosensing Techniques MeSH
• Cell Nucleus genetics   metabolism   MeSH
• DNA chemistry   MeSH
• Fluorescent Dyes chemistry   MeSH
• HeLa Cells MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Nucleotide Motifs MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Fluorescent Dyes MeSH

Telomeres are nucleoprotein structures that distinguish native chromosomal ends from double-stranded breaks. They are maintained by telomerase that adds short G-rich telomeric repeats at chromosomal ends in most eukaryotes and determines the TnAmGo sequence of canonical telomeres. We employed an experimental approach that was based on detection of repeats added by telomerase to identify the telomere sequence type forming the very ends of chromosomes. Our previous studies that focused on the algal order Chlamydomonadales revealed several changes in telomere motifs that were consistent with the phylogeny and supported the concept of the Arabidopsis-type sequence being the ancestral telomeric motif for green algae. In addition to previously described independent transitions to the Chlamydomonas-type sequence, we report that the ancestral telomeric motif was replaced by the human-type sequence in the majority of algal species grouped within a higher order clade, Caudivolvoxa. The Arabidopsis-type sequence was apparently retained in the Polytominia clade. Regarding the telomere sequence, the Chlorogonia clade within Caudivolvoxa bifurcates into two groups, one with the human-type sequence and the other group with the Arabidopsis-type sequence that is solely formed by the Chlorogonium species. This suggests that reversion to the Arabidopsis-type telomeric motif occurred in the common ancestral Chlorogonium species. The human-type sequence is also synthesized by telomerases of algal strains from Arenicolinia, Dunaliellinia and Stephanosphaerinia, except a distinct subclade within Stephanosphaerinia, where telomerase activity was not detected and a change to an unidentified telomeric motif might arise. We discuss plausible reasons why changes in telomeric motifs were tolerated during evolution of green algae.

• Keywords
• 18S rDNA phylogeny, Green algae, TRAP, Telomerase activity, Telomere evolution,
• MeSH
• Amino Acid Motifs genetics   MeSH
• Phylogeny MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Telomerase genetics   MeSH
• Telomere genetics   MeSH
• Volvocida genetics   MeSH
• Telomere Shortening genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH
• RNA, Ribosomal, 18S MeSH
• Telomerase MeSH

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ∼130 μs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-μs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include non-specific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g. structure the propeller loops.

• MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Cations chemistry   MeSH
• Humans MeSH
• Oxytricha genetics   MeSH
• Molecular Dynamics Simulation * MeSH
• Telomere chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Cations MeSH