Dependence of the torsional rigidity of DNA on base composition

The Escherichia coli phage 434 repressor binds as a dimer to the operator of the DNA helix. Although the centre of the operator is not in contact with protein, the repressor binding affinity can be reduced at least 50-fold by changing the sequence there: operators with A.T base pairs near their centre bind the repressor more strongly than do operators with G.C base pairs at the same positions. To explain these observations, it has been proposed that the base composition at the centre of the operator affects the affinity of the operator for repressor by altering the ease with which operator DNA can undergo the torsional deformation necessary for complex formation. In this model, the variation in binding affinity would require the torsion constant to have specific values and to change in a sequence-dependent manner. We have now measured torsion constants for DNAs with widely different base compositions. Our results indicate that the torsion constants depend only slightly on the overall composition, and firmly delimit the range of values for each. Even the upper-limit values are much too small to account for the observed changes in affinity of the 434 repressor. These results rule out simple models that rely on substantial generic differences in torsion constant between A.T-rich sequences and G.C-rich sequences, although they do not rule out the possibility of particular sequences having abnormal torsion constants.     

High-resolution structure of a DNA helix containing mismatched base pairs

The concept of complementary base pairing, integral to the double-helical structure of DNA, provides an effective and elegant mechanism for the faithful transmission of genetic information. Implicit in this model, however, is the potential for incorporating non-complementary base pairs (mismatches) during replication or subsequently, for example, during genetic recombination. As such errors are usually damaging to the organism, they are generally detected and repaired. Occasionally, however, the propagation of erroneous copies of the genome confers a selective advantage, leading to genetic variation and evolutionary change. An understanding of the nature of base-pair mismatches at a molecular level, and the effect of incorporation of such errors on the secondary structure of DNA is thus of fundamental importance. We now report the first single-crystal X-ray analysis of a DNA fragment, d(GGGGCTCC), which contains two non-complementary G X T base pairs, and discuss the implications of the results for the in vivo recognition of base-pair mismatches.     

Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs

The crystal structure of the RNA dodecamer duplex (r-GGACUUCGGUCC)2 has been determined. The dodecamers stack end-to-end in the crystal, simulating infinite A-form helices with only a break in the phosphodiester chain. These infinite helices are held together in the crystal by hydrogen bonding between ribose hydroxyl groups and a variety of donors and acceptors. The four noncomplementary nucleotides in the middle of the sequence did not form an internal loop, but rather a highly regular double-helix incorporating the non-Watson-Crick base pairs, G.U and U.C. This is the first direct observation of a U.C (or T.C) base pair in a crystal structure. The U.C pairs each form only a single base-base hydrogen bond, but are stabilized by a water molecule which bridges between the ring nitrogens and by four waters in the major groove which link the bases and phosphates. The lack of distortion introduced in the double helix by the U.C mismatch may explain its low efficiency of repair in DNA. The G.U wobble pair is also stabilized by a minor-groove water which bridges between the unpaired guanine amino and the ribose hydroxyl of the uracil. This structure emphasizes the importance of specific hydrogen bonding between not only the nucleotide bases, but also the ribose hydroxyls, phosphate oxygens and tightly bound waters in stabilization of the intramolecular and intermolecular structures of double helical RNA.     

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.     

The conformation of the DNA double helix in the crystal is dependent on its environment

Studies of the crystal structures of more than 30 synthetic DNA fragments have provided structural information about three basic forms of the double helix: A-, B- and Z-form DNA. These studies have demonstrated that the DNA double helix adopts a highly variable structure which is related to its base sequence. The extent to which such observed structures are influenced by the crystalline environment can be found by studying the same molecule in different crystalline forms. We have recently crystallized one particular oligomer in various crystal forms. Here we report the results of structural analyses of the different crystal structures and demonstrate that the DNA double helix can adopt a range of conformations in the crystalline state depending on hydration, molecular packing and temperature. These results have implications on our understanding of the influence of the environment on DNA structure, and on the modes of DNA recognition by proteins.     

Salen-金属配合物与DNA中碱基的相互作用

参照文献方法,合成了N,N’-双(2-羟基-1-萘甲醛)缩邻苯二胺、乙二胺和1,3-丙二胺的3种Salen配体及其与Mn(Ⅲ)、Co(Ⅲ)形成的6种配合物,探究了它们对碱基的荧光猝灭效应.结果表明,对于同一碱基来说,Mn(Ⅲ)配合物比Co(Ⅲ)更易使碱基发生荧光猝灭;同种配合物与不同的碱基作用时,其猝灭强度大小顺序为胞嘧啶腺嘌呤鸟嘌呤,呈现出配合物对碱基作用的选择性;配合物的结构对荧光的猝灭也有影响.     

Local destabilisation of a DNA double helix by a T--T wobble pair.

Nuclear magnetic resonance is a technique which permits direct observation of the Waton--Click hydrogen-bonded ring imino protons (guanine N1H and thymine N3H). As the formation and disruption of hydrogen bonds of double-helical RNA and DNA structures are key events during various biological processes, NMR thus provides a useful tool for studying the fluctuational mobility of the individual base pairs. Indeed, several NMR studies of oligo- and polynucleotides have been carried out to probe the structure and dynamics of nucleic acids in solution (for a review see ref. 1). The present study constitutes the first part of our attempt to assess the influence of non-complementary base pairs on the stability of nucleic acid double helices. We report the spectral assignment and temperature-dependent NMR profiles of the hydrogen-bonded imino protons of the two DNA fragments shown in Fig. 1. The assignment is based solely on experimental grounds using the principle of chemical modification. It will be demonstrated that the introduction of a non-complementary (wobble) base pair in a DNA duplex introduces an extra melting site in addition to the sequential melting which starts with the terminal base pairs in the double helix structure.     

Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.     

Analysis of one million base pairs of Neanderthal DNA

Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. We have identified a 38,000-year-old Neanderthal fossil that is exceptionally free of contamination from modern human DNA. Direct high-throughput sequencing of a DNA extract from this fossil has thus far yielded over one million base pairs of hominoid nuclear DNA sequences. Comparison with the human and chimpanzee genomes reveals that modern human and Neanderthal DNA sequences diverged on average about 500,000 years ago. Existing technology and fossil resources are now sufficient to initiate a Neanderthal genome-sequencing effort.