Preferred positions of AA and TT dinucleotides in aligned nucleosomal DNA sequences.

Multiple alignment of 118 nucleosomal DNA sequences by maximizing simultaneously match of AA dinucleotides and match of TT dinucleotides results in a pattern of the dinucleotide distributions which is characteristic of the nucleosomal DNA sequences. The AA dinucleotides are found to be distributed symmetrically relative to the TT dinucleotide distribution, around the middle point of the nucleosomal DNA sequence. The distances between major peaks of the distributions are multiples of about 10.4 bases. The peaks of the TT distribution are shifted by 6 bases downstream from the peaks of the AA distribution.     

Stability of DNA duplexes containing GG, CC, AA, and TT mismatches

We employed salt-dependent differential scanning calorimetric measurements to characterize the stability of six oligomeric DNA duplexes (5'-GCCGGAXTGCCGG-3'/5'-CCGGCAYTCCGGC-3') that contain in the central XY position the GC, AT, GG, CC, AA, or TT base pair. The heat-induced helix-to-coil transitions of all the duplexes are associated with positive changes in heat capacity, DeltaC(p), ranging from 0.43 to 0.53 kcal/mol. Positive values of DeltaC(p) result in strong temperature dependences of changes in enthalpy, DeltaH degrees, and entropy, DeltaS degrees , accompanying duplex melting and cause melting free energies, DeltaG degrees, to exhibit characteristically curved shapes. These observations suggest that DeltaC(p) needs to be carefully taken into account when the parameters of duplex stability are extrapolated to temperatures distant from the transition temperature, T(M). Comparison of the calorimetric and van't Hoff enthalpies revealed that none of the duplexes studied in this work exhibits two-state melting. Within the context of the central AXT/TYA triplet, the thermal and thermodynamic stabilities of the duplexes in question change in the following order: GC > AT > GG > AA approximately TT > CC. Our estimates revealed that the thermodynamic impact of the GG, AA, and TT mismatches is confined within the central triplet. In contrast, the thermodynamic impact of the CC mismatch propagates into the adjacent helix domains and may involve 7-9 bp. We discuss implications of our results for understanding the origins of initial recognition of mismatched DNA sites by enzymes of the DNA repair machinery.     

Curved DNA

A priori considerations and the concept of the sequence-dependent local curving of the DNA axis. Experimental evidence: electric dichroism (relaxation time measurements); anomalous electrophoretic mobility and gel-filtration of some restriction fragments of DNA; one-sided binding of the nucleosomal DNA to the mica surface. Theoretical predictions concerning the nucleotide sequences of the curved DNA. Discovery of the dinucleotide periodicity in the chromatin DNA. The sequence periodicity as a tool for mapping of the nucleosomes along the sequences. Preferential binding of the histone octamers to the curved pieces of DNA--sequence analysis predictions and comparison with experiments: Theoretical and experimental estimates of the tilt and roll angles for different combinations of the neighboring base-pairs. Inherent sequence-dependent curvature and apparent persistence length of DNA.     

Experimental evaluation of the Liu–Beveridge dinucleotide step model of DNA structure

Methods for predicting DNA curvature have many possible applications. Dinucleotide step models describe DNA shape by characterization of helical twist, deflection angles and the direction of deflection for nearest neighbor base pairs. Liu and Beveridge have extended previous applications of dinucleotide step models with the development and qualitative validation of a predictive method for sequence-dependent DNA curvature (the LB model). We tested whether the LB model accurately predicts experimentally deduced curvature angles and helical repeat parameters for DNA sequences not in its training set, particularly when challenged with quantitative data and subtle sequence phasings. We examined a series of 17 well-characterized DNA sequences to compare electrophoretic and computational results. The LB model is superior to two other models in the prediction of helical repeat parameters. We observed a strong linear correlation between curvature magnitudes predicted using the LB model and those determined by electrophoretic ligation ladder experiments, although the LB model somewhat underestimated apparent curvature. With longer electrophoretic phasing probes the LB model slightly overestimated gel mobility anomalies, with modest deviations in predicted helical repeat parameters. Overall, our analyses suggest that the LB model provides reasonably accurate predictions for the electrophoretic behavior of DNA.     

Structural bistability of repetitive DNA elements featuring CA/TG dinucleotide steps and mode of evolution of satellite DNA.

Satellite DNA sequences are known to be important components required for the construction of centromeres and are common to all higher eukaryotes. Nevertheless, their nucleotide sequences vary significantly, even in evolutionarily related species. In order to elucidate how the nucleotide sequences define the conformational character of centromeric satellite DNA, an evolutionary path toward repetitive units has been hypothesized. In that context, the DNA conformation of fish satellite DNA was evaluated in two ways: the organization of subrepeats and sequence characteristics were compared, and the differences in stacking energies between A-helix and B-helix and the sequence-dependent bendability of the helices were evaluated. Our findings suggest that the monomeric units making up currently observed repetitive sequences have evolved through stepwise amplification of shorter, ancestral sequences by increasing the length of the units. In addition, we suggest that potentially key sequences required for DNA amplification comprise highly flexible structures. Thus flexibility of the DNA structure may be a primary prerequisite for DNA amplification.     

An endonucleolytic activity of nuclease TT1 specific for superhelical DNA.

Highly purified nuclease TT1 from T. thermophilus HB8 acts on a linear single- and double-stranded DNA as an exonuclease and produces 5'-mononucleotides either from the 5'- or 3'-terminus. It was found that the enzyme also possesses an endonuclease activity specific for superhelical (form I) and single-stranded circular DNA. Form I of various kinds of DNA (phi X174, PM2, Co1E1 and RF 1010 etc.) is nicked to yield first relaxed circles (form II) and then nicked at the opposite site to yield unit length linear DNA (form III), which is subsequently hydrolyzed from the 5'- or 3'-terminus. A single cleavage of the form I of phi X174 DNA seemed to occur at a limited number of unique sites. Both endonuclease and the known exonuclease activities co-migrate on polyacrylmide gels, show the same pH and temperature optima, are stimulated by Mg2+ and are inactivated by EDTA similarly.     

A refined prediction method for gel retardation of DNA oligonucleotides from dinucleotide step parameters: reconciliation of DNA bending models with crystal structure data

The development and assessment of a prediction method for gel retardation and sequence dependent curvature of DNA based on dinulcleotide step parameters are described. The method is formulated using the Babcock-Olson equations for base pair step geometry (1) and employs Monte Carlo simulated annealing for parameter optimization against experimental data. The refined base pair step parameters define a stuctural construct which, when the width of observed parameter distributions is taken into account, is consistent with the results of DNA oligonucleotide crystal structures. The predictive power of the method is demonstrated and tested via comparisons with DNA bending data on sets of sequences not included in the training set, including A-tracts with and without periodic helix phasing, phased A4T4 and T4A4 motifs, a sequence with a phased GGGCCC motif, some "unconventional" helix phasing sequences, and three short fragments of kinetoplast DNA from Crithidia fasiculata that exhibit significantly different behavior on non-denaturing polyacrylamide gels. The nature of the structural construct produced by the methodology is discussed with respect to static and dynamic models of structure and representations of bending and bendability. An independent theoretical account of sequence dependent chemical footprinting results is provided. Detailed analysis of sequences with A-tract induced axis bending forms the basis for a critical discussion of the applicability of wedge models,junction models and non A-tract, general sequence models for understanding the origin of DNA curvature at the molecular level.     

Sequence-dependent DNA structure: dinucleotide conformational maps

We have used a computational model to calculate the potential energy surface for dinucleotide steps in double helical DNA as a function of the two principal degrees of freedom, slide and shift. By using a virtual bond to model the constraints imposed by the sugar-phosphate backbone, twist, roll, tilt and rise can be simultaneously optimised for any given values of slide and shift. Thus we have been able to construct complete conformational maps for all step types. For some steps, the maps agree well with experimental data from X-ray crystal structures, but other steps appear to be strongly perturbed by the effects of context (conformational coupling with the neighbouring steps). The optimised values of twist and roll show sequence-dependent variations consistent with the crystal structure data. The conformational maps allow us to construct adiabatic paths, and hence calculate the flexibility of each step with respect to slide and shift. Again the results agree well with the available experimental assignments of flexibility: YR steps, CA/TG and CG, are the most flexible and RR steps, such as AA, the least flexible.     

Curved DNA fragments display retarded elution upon anion exchange HPLC.

Restriction fragments containing established curved regions, such as the pBR322 tn3 region, the phage lambda attP region and the SV40 T-antigen and terminus of replication regions, exhibit systematically retarded elution upon anion exchange based HPLC. Using this method, we were able to detect readily other SV40 curved regions, exhibiting also the polyacrylamide gel electrophoresis retardation anomaly, including several RNA polymerase initiation sites. Unlike gel retardation, HPLC retardation exhibited by curved DNA persists at 55 degrees C and is observed for fragments ranging from 150 bp up to 5 kb. The observed preferential attachment of DNA fragments containing curved DNA to the ionic groups of the column suggests a common dipole character of these regions due to the local accumulation of charges resulting apparently from the compression in the minor groove of curved DNA.     

Circular double-stranded forms of TT virus DNA in the liver

TT virus (TTV) is an unenveloped, circular, and single-stranded DNA virus commonly infecting human beings worldwide. TTV DNAs in paired serum and liver tissues from three viremic individuals were separated by gel electrophoresis and characterized biophysically. TTV DNAs in sera migrated in sizes ranging from 2.0 to 2.5 kb. TTV DNAs in liver tissues, however, migrated at 2.0 to 2.5 kb as well as at 3.5 to 6.1 kb. Both faster- and slower-migrating forms of TTV DNAs in the liver were found to be circular and of the full genomic length of 3.8 kb. TTV DNAs migrating at 2.0 to 2.5 kb, from either serum or liver tissues, were sensitive to S1 nuclease but resistant to restriction endonucleases, and therefore, they were single-stranded. By contrast, TTV DNAs in liver tissues that migrated at 3.5 to 6.1 kb were resistant to S1 nuclease. They migrated at 3.7 to 4.0 kb after digestion with EcoRI, which suggests that they represent circular, double-stranded replicative intermediates of TTV. When TTV DNAs were subjected to strand-specific primer extension and then amplified by PCR with internal primers, those in serum were found to be minus-stranded DNAs while those in liver tissues were found to be a mixture of plus- and minus-stranded DNAs. These results suggest that TTV replicates in the liver via a circular double-stranded DNA.     

Intercalation of an acridine-peptide drug in an AA/TT base step in the crystal structure of [d(CGCGAATTCGCG)](2) with six duplexes and seven Mg(2+) ions in the asymmetric unit

We present the crystal structure of an acridine drug derivatized at carbon 9, [N(alpha)-(9-acridinoyl)-tetraarginine], intercalated within the dodecamer [d(CGCGAATTCGCG)](2). The presence of a lateral chain at the central carbon 9 atom differentiates this compound from most acridine drugs hitherto studied, which are usually derivatized at carbon 4. The DNA:drug interaction we observe differs from that observed in previous studies, which primarily involves shorter, mainly hexameric sequences, in two important regards: the acridine intercalates within an AA/TT base step, rather than within a CG/CG base step; and the binding site is located at the center of the sequence, rather than at one end of the duplex. In addition, we observe a novel crystal packing arrangement, with six dodecamer duplexes and seven hydrated magnesium ions in the asymmetric unit of a large (66.5 x 68.4 x 77.4 A(3)) unit cell in space group P2(1)2(1)2(1). The duplexes are organized in layers parallel to the ab plane, with consecutive layers crossing each other at right angles.     

Deletion of a conserved dinucleotide inhibits the second step of group II intron splicing

Few point mutations have been described that specifically inhibit the second step of group II intron splicing. Furthermore, the effects of such mutations are typically not apparent unless the mutations are studied in the context of a substrate that harbors a very short 5' exon. Truncation of the 5' exon slows the second step of splicing. Once the second step has been slowed, the effects of point mutations can be seen. We report the unexpected observation that the deletion of a conserved GA dinucleotide dramatically inhibits the second step of splicing, even when the mutation is studied in the context of a full-length substrate. In contrast, we find that this mutation does not significantly affect the first step of splicing, unless the mutation is studied in combination with a second point mutation that is known to inhibit the first step. Even in that context, the effect of the GA deletion mutation on the first step is modest. These observations, together with the inferred location of the GA dinucleotide in the three-dimensional structure of the intron, suggest that this dinucleotide plays a particularly important role in the second step of splicing.     

Native human TATA-binding protein simultaneously binds and bends promoter DNA without a slow isomerization step or TFIIB requirement

The association of TATA-binding protein (TBP) with promoter DNA is central to the initiation and regulation of eukaryotic protein synthesis. Our laboratory has previously conducted detailed investigations of this interaction using yeast TBP and seven consensus and variant TATA sequences. We have now investigated this key interaction using human TBP and the TATA sequence from the adenovirus major late promoter (AdMLP). Recombinant native human protein was used together with fluorescently labeled DNA, allowing real time data acquisition in solution. We find that the wild-type hTBP-DNAAdMLP reaction is characterized by high affinity (Kd < or = 5 nm), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent approximately 100 degrees. These data allow, for the first time, a direct comparison of the reactions of the full-length, native human and yeast TBPs with a consensus promoter, studied under identical conditions. The general reaction characteristics are similar for the human and yeast proteins, although the details differ and the hTBPwt-induced bend is more severe. This directly measured hTBPwt-DNAAdMLP interaction differs fundamentally from a recently published hTBPwt-DNAAdMLP model characterized by low affinity (microM) binding and an unstable complex requiring either a 30-min isomerization or TFIIB to achieve DNA bending. Possible sources of these significant differences are discussed.     

Curved DNA molecules migrate anomalously slowly in free solution

The electrophoretic mobility of a curved DNA restriction fragment taken from the VP1 gene in the SV40 minichromosome has been measured in polyacrylamide gels and free solution, using capillary electrophoresis. The 199 bp restriction fragment has an apparent bend angle of 46 ± 2° located at SV40 sequence position 1922 ± 2 bp [Lu Y.J., Weers B.D. and Stellwagen N. C. (2005) Biophys. J., 88, 1191–1206]. The ‘curvature module’ surrounding the apparent bend center contains five unevenly spaced A- and T-tracts, which are responsible for the observed curvature. The parent 199 bp fragment and sequence mutants containing at least one A-tract in the curvature module migrate anomalously slowly in free solution, as well as in polyacrylamide gels. Hence, the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due in part to their anomalously slow mobilities in free solution. Analysis of the gel and free solution mobility decrements indicates that each A- or T-tract contributes independently, but not equally, to the curvature of the 199 bp fragment and its A-tract mutants. The relative contribution of each A- or T-tract to the observed curvature depends on its spacing with respect to the first A-tract in the curvature module.     

Apoptotic nuclear morphological change without DNA fragmentation.

Apoptosis is characterized morphologically by condensation and fragmentation of nuclei and cells and biochemically by fragmentation of chromosomal DNA into nucleosomal units [1]. CAD, also known as CPAN or DFF-40, is a DNase that can be activated by caspases [2] [3] [4] [5] [6]. CAD is complexed with its inhibitor, ICAD, in growing, non-apoptotic cells [2] [7]. Caspases that are activated by apoptotic stimuli [8] cleave ICAD. CAD, thus released from ICAD, digests chromosomal DNA into nucleosomal units [2] [3]. Here, we examine whether nuclear morphological changes induced by apoptotic stimuli are caused by the degradation of chromosomal DNA. Human T-cell lymphoma Jurkat cells, as well as their transformants expressing caspase-resistant ICAD, were treated with staurosporine. The chromosomal DNA in Jurkat cells underwent fragmentation into nucleosomal units, which was preceded by large-scale chromatin fragmentation (50-200 kb). The chromosomal DNA in cells expressing caspase-resistant ICAD remained intact after treatment with staurosporine but their chromatin condensed as found in parental Jurkat cells. These results indicate that large-scale chromatin fragmentation and nucleosomal DNA fragmentation are caused by an ICAD-inhibitable DNase, most probably CAD, whereas chromatin condensation during apoptosis is controlled, at least in part, independently from the degradation of chromosomal DNA.