Quadruplex structure of Oxytricha telomeric DNA oligonucleotides.

The telomeres of most eukaryotes contain a repeating G-rich sequence with the consensus d(T/A)1-4G1-8, of which 12-16 bases form a 3' single-strand overhang beyond the telomeric duplex. It has been proposed that these G-rich oligonucleotides associate to form four-stranded structures from one, two or four individual strands and that these structures may be relevant in vivo. The proposed structures contain Hoogsteen base-paired G-quartets, precedent for which has been in the literature for many years. Here we use 1H NMR spectroscopy to study the conformations of the DNA oligonucleotides d(G4T4G4) (Oxy-1.5) and d(G4T4G4T4G4T4G4) (Oxy-3.5) which contain the Oxytricha telomere repeat (T4G4). We find that these molecules fold to form a symmetrical bimolecular and an intramolecular quadruplex, respectively. Both structures have four G-quartets formed from nucleotides that are alternately syn and anti along each strand. This arrangement differs from earlier models in which the strands are alternately all syn or all anti. The T4 loops in Oxy-1.5 are on opposite ends of the quadruplex and loop diagonally across the G-quartet, resulting in adjacent strands being alternately parallel and antiparallel.     

A sodium-potassium switch in the formation of four-stranded G4-DNA

Single-stranded complex guanine-rich DNA sequences from chromosomal telomeres and elsewhere can associate to form stable parallel four-stranded structures termed G4-DNA by a process that is anomalously dependent on the particular alkali metal cation that is present. The anomaly, which is not found in the formation of G4-DNA by oligonucleotides containing short, single runs of three or more guanines, is caused by potassium cations excessively stabilizing fold-back intermediate structures, or pathway by-products.     

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.     

利用过氧化物模拟酶催化化学发光法检测钾离子

K+存在时,富含鸟嘌呤(Guanine)的特定序列单链DNA会发生构象变化形成G-四联体结构,从而能够与氯化血红素(hemin)结合生成具有过氧化物酶活性的模拟酶,对鲁米诺-H2O2化学发光体系具有强的催化作用.据此,提出了利用G-四联体-hemin-鲁米诺-H2O2化学发光体系测定K+的新方法.探讨了DNA浓度,he...     

Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops

The telomeric ends of eukaryotic chromosomes are composed of simple repeating sequences in which one DNA strand contains short tracts of guanine residues alternating with short tracts of A/T-rich sequences. The guanine-rich strand is always oriented in a 5'-3' direction towards the end of the chromosome and is extended to produce a 3' overhang of about two repeating units in species where the telomeric terminus is known. This overhang has been implicated in the formation of several unusual intra-and intermolecular DNA structures, although none of these structures has been characterized fully. We now report that oligonucleotides encoding Tetrahymena telomeres dimerize to form stable complexes in solution. This salt-dependent dimerization is mediated entirely by the 3'-terminal telomeric overhang (TT-GGGGTTGGGG) and produces complexes in which the N7 position of every guanine in the overhangs is chemically inaccessible. We therefore propose that telomeric DNA dimerizes by hydrogen bonding between two intramolecular hairpin loops, to form antiparallel quadruplexes containing cyclic guanine base tetrads. These novel hairpin dimers may be important in telomere association and recombination and could also provide a general mechanism for pairing two double helices in other recombinational processes.     

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.     

A role for branchpoints in splicing in vivo

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.     

Molecular evolution of scorpion a-toxins----Accelerated substitutions and functional divergence

Scorpion a-toxins are a family of toxic proteins with similar scaffold, but possess divergent pharmacological properties. Analysis of cDNA sequences reveals that the numbers of nucleotide substitutions per site ( K ) for 5' and 3' UTRs are smaller than those per synonymous site ( Ks) for the mature peptide-coding sequences, whereas the numbers of nucleotide substitutions per nonsynonymous site (-Ka) are close to or larger than Ks values for relevant pairs of cDNAs. These results, together with phylogenetic analysis, indicate that scorpion a-toxins have evolved by accelerated substitutions in the mature toxin regions. In addition, the 15 amino acids, absolutely conserved in all the scorpion a-toxins described so far. are mostly located in molecular interior, which may be involved in structural constraints for stabilizing the CSa(3 fold in evolution of these molecules. Four hot spot mutation sites in the molecular surface are found to distribute in the putative functional regions of a-toxins. suggesting that positive Darwinian selection drives the accelerated evolution of scorpion a-toxins. These findings reasonably explain the relationship between three-dimensional structure conservation and functional divergence of scorpion a-toxins and are of important value in guiding us in our engineering experiments to obtain higher affinity ligands to Na+ channels.     

Inhibition of telomerase by G-quartet DNA structures

The ends or telomeres of the linear chromosomes of eukaryotes are composed of tandem repeats of short DNA sequences, one strand being rich in guanine (G strand) and the complementary strand in cytosine. Telomere synthesis involves the addition of telomeric repeats to the G strand by telomere terminal transferase (telomerase). Telomeric G-strand DNAs from a variety of organisms adopt compact structures, the most stable of which is explained by the formation of G-quartets. Here we investigate the capacity of the different folded forms of telomeric DNA to serve as primers for the Oxytricha nova telomerase in vitro. Formation of the K(+)-stabilized G-quartet structure in a primer inhibits its use by telomerase. Furthermore, the octanucleotide T4G4, which does not fold, is a better primer than (T4G4)2, which can form a foldback structure. We conclude that telomerase does not require any folding of its DNA primer. Folding of telomeric DNA into G-quartet structures seems to influence the extent of telomere elongation in vitro and might therefore act as a negative regulator of elongation in vivo.     

Folding-driven synthesis of oligomers.

The biological function of biomacromolecules such as DNA and enzymes depends on their ability to perform and control molecular association, catalysis, self-replication or other chemical processes. In the case of proteins in particular, the dependence of these functions on the three-dimensional protein conformation is long known and has inspired the development of synthetic oligomers and polymers with the capacity to fold in a controlled manner, but it remains challenging to design these so-called 'foldamers' so that they are capable of inducing or controlling chemical processes and interactions. Here we show that the stability gained from folding can be used to control the synthesis of oligomers from short chain segments reversibly ligated through an imine metathesis reaction. That is, folding shifts the ligation equilibrium in favour of conformationally ordered sequences, so that oligomers having the most stable solution structures form preferentially. Crystallization has previously been used to shift an equilibrium in order to indirectly influence the synthesis of small molecules, but the present approach to selectively prepare macromolecules with stable conformations directly connects folding and synthesis, emphasizing molecular function rather than structure in polymer synthesis.     

R+-SiF3(R=Li,Na,K)分子构型及稳定性的理论研究

在B3LYP/6-311 G(2d,2p)理论水平上对R -SiF3(R=Li,Na,K)各构型进行结构优化,NBO原子电荷分析用NPA程序.用同样的理论水平上对R -SiF3(R=Li,Na,K)进行零点能、总能、焓、相对能量计算和碱金属离子亲和能计算,计算出能量结合频率分析确定配合物构型的稳定性.优化的几何构型、NBO分析表明R (R=Li,Na,K)和SiF3形成的是离子偶极型配合物.Li ,Na ,K 形成最稳定分子的亲和能分别是90.46KJ/mol、57.52KJ/mol和37.83KJ/mol.足够大的锂离子亲和能表明锂离子吸附SiF3的配合物能被锂离子吸附质谱仪探测到.     

Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region.

Selenocysteine is incorporated cotranslationally at UGA codons, normally read as stop codons, in several bacterial proteins and in the mammalian proteins glutathione peroxidase (GPX), selenoprotein P and Type I iodothyronine 5' deiodinase (5'DI). Previous analyses in bacteria have suggested that a stem-loop structure involving the UGA codon and adjacent sequences is necessary and sufficient for selenocysteine incorporation into formate dehydrogenase and glycine reductase. We used the recently cloned 5'DI to investigate selenoprotein synthesis in eukaryotes. We show that successful incorporation of selenocysteine into this enzyme requires a specific 3' untranslated (3'ut) segment of about 200 nucleotides, which is found in both rat and human 5'DI messenger RNAs. These sequences are not required for expression of a cysteine-mutant deiodinase. Although there is little primary sequence similarity between the 3'ut regions of these mRNAs and those encoding GPX, the 3'ut sequences of rat GPX can substitute for the 5'DI sequences in directing selenocysteine insertion. Computer analyses predict similar stem-loop structures in the 3'ut regions of the 5'DI and GPX mRNAs. Limited mutations in these structures reduce or eliminate their capacity to permit 5'DI translation. These results identify a 'selenocysteine-insertion sequence' motif in the 3'ut region of these mRNAs that is essential for successful translation of 5'DI, presumably GPX, and possibly other eukaryotic selenocysteine-containing proteins.     

Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides

Homopyrimidine oligonucleotides bind to homopurine-homopyrimidine sequences of duplex DNA forming a local triple helix. This binding can be demonstrated either directly by a footprinting technique, gel assays, or indirectly by inducing irreversible reactions in the target sequence, such as photocrosslinking or cleavage. Binding occurs in the major groove with the homopyrimidine oligonucleotide orientated parallel to the homopurine strand. Thymine and protonated cytosine in the oligonucleotide form Hoogsteen-type hydrogen bonds with A.T and G.C Watson-Crick base pairs, respectively. Here we report that an 11-residue homopyrimidine oligonucleotide covalently attached to an ellipticine derivative by its 3' phosphate photo-induces cleavage of the two strands of a target homopurine--homopyrimidine sequence. To our knowledge, this is the first reported case of a sequence-specific artificial photoendonuclease. In addition we show that a strong binding site for a free ellipticine derivative is induced at the junction between the triplex and duplex structures on the 5' side of the bound oligonucleotide. On irradiation, cleavage is observed on both strands of DNA. This opens new possibilities for inducing irreversible reactions on DNA at specific sites by the synergistic action of a triple helix-forming oligonucleotide and an intercalating agent.     

Cloning of human telomeres by complementation in yeast

Telomeres confer stability on chromosomes by protecting them from degradation and recombination and by allowing complete replication of the end. They are genetically important as they define the ends of the linkage map. Telomeres of lower eukaryotes contain short repeats consisting of a G-rich and a C-rich strand, the G-rich strand running 5'-3' towards the telomere and extending at the end. Telomeres of human chromosomes share characteristics with those of lower eukaryotes including sequence similarity as detected by cross-hybridization. Telomeric repeats from many organisms can provide telomere function in yeast. Here we describe a modified yeast artificial chromosome (YAC) vector with only one telomere which we used to clone human telomeres by complementation in yeast. YACs containing human telomeres were identified by hydridization to an oligonucleotide of the trypanosome telomeric repeat. A subcloned human fragment from one such YAC is immediately subtelomeric on at least one human chromosome.     

Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase

Telomeres define the ends of chromosomes; they consist of short tandemly repeated DNA sequences loosely conserved in eukaryotes (G1-8(T/A)1-4). Telomerase is a ribonucleoprotein which, in vitro, recognizes a single-stranded G-rich telomere primer and adds multiple telomeric repeats to its 3' end by using a template in the RNA moiety. In conjunction with other components, telomerase may balance the loss of telomeric repeats due to DNA replication. Another role of telomerase may be the de novo formation of telomeres. In eukaryotes like Tetrahymena, this process is an integral part of the formation of macronuclear chromosomes. In other eukaryotes this process stabilizes broken chromosomes. A case of human alpha-thalassaemia is caused by a truncation of chromosome 16 that has been healed by the addition of telomeric repeats (TTAGGG)n. Using an in vitro assay, I show here that human telomerase correctly recognizes the chromosome 16 breakpoint sequence and adds (TTAGGG)n repeats. The DNA sequence requirements are minimal and seem to define two modes of DNA recognition by telomerase.     

Stable RNA hairpins in 88 coding regions of human mRNA

RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R＝G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.     

平面的BC_nN(n=1～5)团簇的结构和稳定性

在CCSD(T)/6-311+G(d)∥B3LYP/6-311+G(d)理论水平上系统研究了平面BCnN(n=1～5)团簇的几何结构和稳定性。BCnN(n=1～5)团簇的最低能量结构是线形结构。在BCnN(n=1～5)团簇的较低能量结构中,线形结构比环状结构以及含有支链的环状结构在能量上更有利。BCnN(n=1～5)团簇的最低能量结构的平均成键能、成键能增量、垂直电离能和垂直亲和能的研究结果显示,异构体BCN、BC3B和BC5N比它们相邻的团簇更稳定。     

A hypersensitive site in hsp70 chromatin requires adjacent not internal DNA sequence

Nuclease-hypersensitive sites in chromatin exist at the 5' side of many eukaryotic genes. To gain some understanding of the molecular basis of these hypersensitive sites, we have now examined the pair of sites upstream of the Drosophila hsp70 gene in a series of plasmids that contain deletions in the hypersensitive region and have been transformed into yeast cells. Hypersensitive sites 5' to a Drosophila hsp70 gene are preserved when this gene is introduced into yeast by transformation. We find that a yeast strain containing a plasmid in which the deletion extends through the first hypersensitive site still displays the normal pair of hypersensitive sites, so DNA sequences over which the first hypersensitive site is centred are not required for hypersensitivity at this position and the site can form over a foreign DNA sequence juxtaposed against this deletion end point. Deletions progressing further into the region bracketed by the pair of 5' hypersensitive sites eliminate the first hypersensitive site and alter the downstream site. We propose that the hypersensitive sites are generated through the binding of a protein that renders flanking sequences more accessible to nucleases, perhaps by preventing normal chromatin packaging.