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

Guanine quadruplexes (G4s) are non-canonical nucleic acids structures common in important genomic regions. Parallel-stranded G4 folds are the most abundant, but their folding mechanism is not fully understood. Recent research highlighted that G4 DNA molecules fold via kinetic partitioning mechanism dominated by competition amongst diverse long-living G4 folds. The role of other intermediate species such as parallel G-triplexes and G-hairpins in the folding process has been a matter of debate. Here, we use standard and enhanced-sampling molecular dynamics simulations (total length of ∼0.9 ms) to study these potential folding intermediates. We suggest that parallel G-triplex per se is rather an unstable species that is in local equilibrium with a broad ensemble of triplex-like structures. The equilibrium is shifted to well-structured G-triplex by stacked aromatic ligand and to a lesser extent by flanking duplexes or nucleotides. Next, we study propeller loop formation in GGGAGGGAGGG, GGGAGGG and GGGTTAGGG sequences. We identify multiple folding pathways from different unfolded and misfolded structures leading towards an ensemble of intermediates called cross-like structures (cross-hairpins), thus providing atomistic level of description of the single-molecule folding events. In summary, the parallel G-triplex is a possible, but not mandatory short-living (transitory) intermediate in the folding of parallel-stranded G4.

• MeSH
• DNA chemistry   genetics   metabolism   MeSH
• G-Quadruplexes * MeSH
• Guanine chemistry   metabolism   MeSH
• DNA, Single-Stranded chemistry   genetics   metabolism   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Base Sequence MeSH
• Molecular Dynamics Simulation * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Guanine MeSH
• DNA, Single-Stranded MeSH