Crystal structure of parallel quadruplexes from human telomeric DNA

Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, are fundamental in protecting the cell from recombination and degradation. Disruption of telomere maintenance leads to eventual cell death, which can be exploited for therapeutic intervention in cancer. Telomeric DNA sequences can form four-stranded (quadruplex) structures, which may be involved in the structure of telomere ends. Here we describe the crystal structure of a quadruplex formed from four consecutive human telomeric DNA repeats and grown at a K(+) concentration that approximates its intracellular concentration. K(+) ions are observed in the structure. The folding and appearance of the DNA in this intramolecular quadruplex is fundamentally different from the published Na(+)-containing quadruplex structures. All four DNA strands are parallel, with the three linking trinucleotide loops positioned on the exterior of the quadruplex core, in a propeller-like arrangement. The adenine in each TTA linking trinucleotide loop is swung back so that it intercalates between the two thymines. This DNA structure suggests a straightforward path for telomere folding and unfolding, as well as ways in which it can recognize telomere-associated proteins.     

DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA

Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions. The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals. Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation. Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown. Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S. pombe Pot1p complexed with ssDNA. The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding. The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA. Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups. The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus.     

An unusual DNA structure detected in a telomeric sequence under superhelical stress and at low pH

Telomeric sequences of DNA, which are found at the ends of linear chromosomes, have been attracting attention as potential sites for the formation of unusual DNA structures. They consist of (GnTm) or (GnATm) motifs (n greater than or equal to m) and, in the single-stranded state, form hairpins stabilized by non-canonical G.G pairs. In the duplex state and under superhelical stress they exhibit hypersensitivity to SI nuclease which by analogy with homopurine-homopyrimidine sequences may reflect the formation of an unusual structure. To determine whether this is the case we have inserted into a plasmid the Tetrahymena telomeric motif (G4T2).(A2C4) and probed it by two-dimensional gel electrophoresis, chemical modification and oligonucleotide binding. Our data demonstrate that, under superhelical stress and at low pH, the insert does indeed adopt a novel DNA conformation. We have concluded that in this structure the C-rich strand forms a hairpin stabilized by non-Watson-Crick base pairs C.C+ and A.A+, whereas the G-rich strand remains unstructured. We term this new DNA structure the (C,A)-hairpin.     

Spectroscopic investigation on the telomeric DNA base sequence repeat

Telomeres are protein-DNA complexes at the terminals of linear chromosomes, which protect chromosomal integrity and maintain cellular replicative capacity. From single-cell organisms to advanced animals and plants, structures and functions of telomeres are both very conservative. In cells of human and vertebral animals, telomeric DNA base sequences all are (TTAGGG)n. In the present work, we have obtained absorption and fluorescence spectra measured from seven synthesized oligonucleotides to simulate the telomeric DNA system and calculated their relative fluorescence quantum yields on which not only telomeric DNA characteristics are predicted but also possibly the shortened telomeric sequences during cell division are implied. Oligonucleotide 5′-TTAGGGTTAGGG holds a low relative fluorescence quantum yield and remarkable excitation energy innerconversion, which tallies with the telomeric sequence of (TTAGGG)n. This result shows that telomeric DNA has a strong non-radiative or innerconvertible capability.     

Circular dichroism spectroscopic studies on structures formed by telomeric DNA sequencesin vitro

Telomere plays an important role in cellular processes, such as cell aging, death and carcinogenisis. Having special sequences, it can form quadruplex structurein vitro. Circular dichroism (CD) spectroscopic studies show that TTAGGG, (TTAGGG)₂ and (TTAGGG)₄ can all form quadruplexin vitro and exist mainly as parallel quadruplex without metal ions. Both K⁺ and Na⁺ can stabilize the tetrameric structure and facilitate the forming of anti-parallel conformation. Furthermore, the conformations of quadruplex can also be affected by sequence length, the nature and concentration of metal ions.     

Circular dichroism spectroscopic studies on structures formed by telomeric DNA sequences in vitro

Telomere plays an important role in cellular processes, such as cell aging, death and carcinogenisis. Having special sequences, it can form quadruplex structure in vitro. Circular dichroism (CD) spectroscopic studies show that TTAGGG, (TTAGGG)2 and (TTAGGG)4 can all form quadruplex in vitro and exist mainly as parallel quadruplex without metal ions. Both K+ and Na+ can stabilize the tetrameric structure and facilitate the forming of anti-parallel conformation. Furthermore, the conformations of quadruplex can also be affected by sequence length, the nature and concentration of metal ions.     

TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA

Maintenance of telomeres requires both DNA replication and telomere 'capping' by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity.     

Crystal structure of an N-terminal fragment of the DNA gyrase B protein.

The crystal structure of an N-terminal fragment of the Escherichia coli DNA gyrase B protein, complexed with a nonhydrolysable ATP analogue, has been solved at 2.5 A resolution. It consists of two domains, both containing novel protein folds. The protein fragment forms a dimer, whose N-terminal domains are responsible for ATP binding and hydrolysis. The C-terminal domains form the sides of a 20 A hole through the protein dimer which may play a role in DNA strand passage during the supercoiling reaction.     

Use of an 125I-labelled DNA ligand to probe DNA structure

It no longer seems likely that DNA molecules in situ have a uniform conformation, represented by the classical B-form helix. For example, recent structural studies have shown that in certain conditions DNA can have a left-handed (so-called Z-form) helix, and have revealed extensive sequence-dependent variations of B-DNA helical parameters. Such sequence-dependent variations in DNA structure can be investigated in solution with reagents that bind to DNA in a conformation-dependent manner, and cut one or both strands of the double-helix at the site of binding, as, for example, has been shown for the endonuclease DNase I3. We describe here a simple way to endow a DNA-binding ligand with the ability to cleave DNA--labelling with 125I. The radiochemical damage associated with 125I decay induces a double-stranded DNA break. Using this technique we have shown that a sequence of four consecutive A X T base pairs is a necessary, but not sufficient, condition for strong binding to DNA of the bis-benzamide Hoechst 33258--presumably the other important factor is the conformation of the double-helix at the site of the (A/T)4 sequence. We suggest 125I-Hoechst 33258 may be a useful new probe of DNA structure.     

X-ray structure of a DNA hairpin molecule

We have solved the crystal structure of a synthetic DNA hexadecanucleotide of sequence: C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G, at 2.1 A resolution, and observed that it adopts a monomeric hairpin configuration with a Z-DNA hexamer stem. In the T4 loop the bases stack with one another and with neighbouring molecules of the crystal, and not with base pairs of their own hexamer stem. Two thymine T10 rings from different molecules stack between the C1-G16 ends of a third and a fourth hairpin helix, in a manner that suggests T-T base 'pairing' and simulates a long, 13-base-pair helix. Although such T-T interactions would not be present in solution, they illustrate a remarkable tendency of thymines for self-association. Purine-purine G-A base pairs are known to exist in the anti-anti conformation with an increase in local helix width; it may be that more serious consideration should be given to the possible existence of pyrimidine-pyrimidine C-T base pairs with decreased local helix width, particularly where several such base pairs occur sequentially.     

A novel DNA computing model based on RecA-mediated triple-stranded DNA structure

The field of DNA computing emerged in 1994 after Adleman’s paper was published. Henceforth,a few scholars solved some noted NP-complete problems in this way. And all these methods of DNA computing are based on conventional Watson-Crick hydrogen bond of doublehelical DNA molecule. In this paper, we show that the triple-stranded DNA structure mediated by RecA protein can be used for solving computational problems. Sequence-specific recognition of double-stranded DNA by oligonucleotide-directed triple helix (triplex) formation is used to carry out the algorithm. We present procedure for the 3-vertex-colorability problems. In our proposed procedure, it is suggested that it is possible to solve more complicated problems with more variables by this model.     

Primary structure and functional expression from complementary DNA of a brain calcium channel.

The primary structure of a voltage-dependent calcium channel from rabbit brain has been deduced by cloning and sequencing the complementary DNA. Calcium channel activity expressed from the cDNA is dramatically increased by coexpression of the alpha 2 and beta subunits, known to be associated with the dihydropyridine receptor. This channel is a high voltage-activated calcium channel that is insensitive both to nifedipine and to omega-conotoxin. We suggest that it is expressed predominantly in cerebellar Purkinje cells and granule cells.