Species-specific patterns of DNA bending and sequence.

Nucleotide sequences in the GenEMBL database were analyzed using strategies designed to reveal species-specific patterns of DNA bending and DNA sequence. The results uncovered striking species-dependent patterns of bending with more variations among individual organisms than between prokaryotes and eukaryotes. The frequency of bent sites in sequences from different bacteria was related to genomic A + T content and this relationship was confirmed by electrophoretic analysis of genomic DNA. However, base composition was not an accurate predictor for DNA bending in eukaryotes. Sequences from C. elegans exhibited the highest frequency of bent sites in the database and the RNA polymerase II locus from the nematode was the most bent gene in GenEMBL. Bent DNA extended throughout most introns and gene flanking segments from C.elegans while exon regions lacked A-tract bending characteristics. Independent evidence for the strong bending character of this genome was provided by electrophoretic studies which revealed that a large number of the fragments from C.elegans DNA exhibited anomalous gel mobilities when compared to genomic fragments from over 20 other organisms. The prevalence of bent sites in this genome enabled us to detect selectively C.elegans sequences in a computer search of the database using as probes C.elegans introns, bending elements, and a 20 nucleotide consensus sequence for bent DNA. This approach was also used to provide additional examples of species-specific sequence patterns in eukaryotes where it was shown that (A) greater than or equal to 10 and (A.T) greater than or equal to 5 tracts are prevalent throughout the untranslated DNA of D.discodium and P.falciparum, respectively. These results provide new insight into the organization of eukaryotic DNA because they show that species-specific patterns of simple sequences are found in introns and in other untranslated regions of the genome.     

Bending of DNA segments with Saccharomyces cerevisiae autonomously replicating sequence activity, isolated from basidiomycete mitochondrial linear plasmids

Summary Previous studies have indicated that DNA bending is a general structural feature of sequences (ARSs) from cellular DNAs of yeasts and nuclear and mitochondrial genomic DNAs of other eukaryotes that are capable of autonomous replication in Saccharomyces cerevisiae. Here we showed that bending activity is also tightly associated with S. cerevisiae ARS function of segments cloned from mitochondrial linear DNA plasmids of the basidiomycetes Pleurotus ostreatus and Lentinus edodes. Two plasmids, designated pLPO2-like (9.4 kb), and pLPO3 (6.6 kb) were isolated from a strain of P. ostreatus. A 1029 by fragment with high-level ARS activity was cloned from pLPO3 and it contained one ARS consensus sequence (A/T)TTTAT(A/G)TTT(A/T) indispensable for activity and seven dispersed ARS consensus-like (10/11 match) sequences. A discrete bent DNA region was found to lie around 500 by upstream from the ARS consensus sequence (T-rich strand). Removal of the bent DNA region impaired ARS function. DNA bending was also implicated in the ARS function associated with a 1430 by fragment containing three consecutive ARS consensus sequences which had been cloned from the L. edodes plasmid pLLE1 (11.0 kb): the three consecutive ARSs responsible for high-level ARS function occurred in, and immediately adjacent to, a bent DNA region. A clear difference exists between the two plasmid-derived ARS fragments with respect to the distance between the bent DNA region and the ARS consensus sequence(s).     

RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.     

Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.     

Attenuation of DNA-protein interactions associated with intrinsic, sequence-dependent DNA curvature.

Inherently curved DNA segments, associated with short runs of adenines, have been identified in many gene regulatory regions, yet their physiological significance remains unknown. The observations reported in this study indicate that intrinsically bent nucleic acid fragments are characterized by substantially attenuated affinities toward DNA-binding proteins involved in structural functions, such as H1 histone and protamine, as well as toward various DNA-modifying enzymes including ligases and exo- and endonucleases. Two mechanisms might be responsible for the altered binding properties. According to the first mechanism, the attenuated binding affinities and the bending represent two independent consequences of the unique structural parameters exhibited by A-tracts. Indeed, analysis of the degradation products obtained upon exposure of the curved sequences to various chemical nucleases points toward the narrowing of the DNA minor groove, a conformational modulation known to characterize A-tracts and to run along the axially-bent motifs, as a potential determinant of the observed binding attenuation. Alternatively, the conformational constraints which result from the stable bending might act to modulate the strength of DNA-protein interactions. Although the factor directly responsible for the altered binding affinities revealed by the bent sequences cannot as yet be conclusively resolved, it is proposed that a reiteration of this specific factor, being either an A-tract or a bend, in phase with the DNA helical repeat acts to amplify the modulation of the binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sequence-directed bent DNA detected in the replication origin of Chlamydomonas reinhardtii chloroplast DNA is important for the replication function

Summary We demonstrated that the 1055 by restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A₄ tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 by subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.     

DNA bending versus DNA end joining activity of HMGB1 protein is modulated in vitro by acetylation

The ability of HMGB1 protein to recognize bent DNA and to induce bending in linear duplex DNA defines HMGB1 as an architectural factor. It has already been demonstrated that the binding affinity of the protein for various bent DNA structures is enhanced upon in vivo acetylation at Lys2. Here we investigate how this modification of HMGB1 affects its ability to bend DNA. We report that the modified protein cannot bend short DNA fragments but, instead, stimulates joining of the same fragments via their ends. The same properties are exhibited in vivo by acetylated HMGB1 lacking its acidic tail. Further, in vitro acetylation of the truncated protein at Lys81 (possible upon tail removal only) restores the protein's bending ability, while the level of stimulation of DNA end joining is strongly reduced. We conclude, therefore, that the ability of HMGB1 to bend DNA or to stimulate end joining is modulated in vitro by acetylation. In an attempt to explain the properties of in vivo-acetylated HMGB1, its complexes with DNA have been analyzed by both protein-DNA cross-linking and atomic force microscopy. Unlike the parental protein, bound mainly within the internal sequences, acetylated HMGB1 binds preferentially to DNA ends. We propose that the loading of acetylated protein on DNA ends accounts for both the failure to bend DNA and the stimulation of DNA end joining.     

The bending of DNA in nucleosomes and its wider implications

The DNA of a nucleosome core particle is wrapped tightly around a histone octamer with approximately 80 base pairs per superhelical turn. Studies of both naturally occurring and reconstituted systems have shown that DNA sequences very often adopt well-defined locations with respect to the octamer. Recent work in this laboratory has provided a structural explanation for this sequence-dependent positioning in terms of the differential flexibility of different sequences and of departures from smooth bending. The 'rules' that are emerging for DNA bendability and, from the results of other workers, on intrinsically bent DNA, are likely to be useful in considering looping and bending of DNA in other processes in which it is thought to be wrapped around a protein core.     

Tet repressor binding induced curvature of tet operator DNA.

Tet repressor dimer binds to two tet operator sites spaced by 30 bp in the Tn10 encoded tet regulatory DNA. The effect of repressor binding on the gel mobility of circular permutated DNA fragments containing either one or both operator sequences is reported. The EcoRI induced bending of DNA is used to compare the results with other protein binding induced structural perturbations of DNA. Tet repressor bends a DNA fragment with a single tet operator to an angle of 42 degrees +/- 7 degrees. The apparent bend angle of DNA fragments containing the tandem tet operator arrangement occupied by two Tet repressor dimers turns out to be 52 degrees +/- 9 degrees. These results are interpreted with respect to the end to end distances of the bent DNA fragments. They indicate that either the intervening tet regulatory DNA between the operators or the bound operator sequences themselves contain additional perturbations from the canonical B-DNA structure. This finding is discussed in the light of previously obtained results from CD, neutron scattering, and electrooptical studies.     

Anisotropic flexibility of DNA depends on the base sequence. Conformation calculations of double-stranded tetranucleotides AAAA:TTTT, (AATT)2, (TTAA)2, GGGG:CCCC, (GGCC)2, (CCGG)2

The bending flexibility of six tetramers was studied in an assumption that they were extended in the both directions by regular double helices. The bends of B-DNA in different directions were considered. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less pronounced than in the perpendicular direction by the order of magnitude. Such an anisotropy is a feature of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5-7 degrees, are in agreement with the experimental value of DNA persistence length. Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove, moreover, they have an equilibrium bend of 6-12 degrees into this groove. The above inequality is caused by the stacking interaction of the bases. The bend in the central dimers is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is unessential, so that DNA remains within the limits of the B-family of forms: namely, when the helical axis is bent by 20 degrees the backbone dihedral angles vary by no more than 15 degrees. The obtained results are in accord with the X-ray structure of B-DNA dodecamer; they further substantiate our earlier model of DNA wrapping in the nucleosome by means of "mini-kinks" separated by a half-pitch of the double helix, i.e. by 5-6 b. p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimensional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in the equilibrium structure of certain DNA fragments. To the four "Calladine rules" two more can be added: the minor-groove steric clash of purines in the YR sequences are avoided by: (1) bending of the helix into the major groove; (2) increasing the distance between the base pairs (stretching the double helix).     

Chromosomal illegitimate recombination in mammalian cells is associated with intrinsically bent DNA elements.

Illegitimate recombination is the most frequent mechanism for chromosomal rearrangements in mammalian cells, yet little is known about this process. Most of the studies to date have looked at the sequences present at illegitimate junctions. These revealed the presence of recurrent DNA motifs, none of which was consistently found. We have undertaken to determine if intrinsic DNA structures such as bent DNA elements could be a major determinant in chromosomal illegitimate recombination. Using a two dimensional electrophoretic assay we found that eight out of eight junctions, resulting from various types of chromosomal rearrangements, had migration behaviour characteristic of DNA containing intrinsically bent DNA elements. In all cases, these occurred within one kilobase of the junctions, and in most cases could be found in both participating DNA segments. We also found that these bent DNA elements were present before the recombination event. When we analysed the frequency of intrinsically bent DNA elements in random chromosomal fragments, we found it to be about one per 11 kilobases. Thus these results suggest that bent DNA is associated with chromosomal illegitimate recombination.     

Detection, sequence patterns and function of unusual DNA structures.

Unusual DNA structures were detected by an electrophoretic procedure in which DNA fragments were separated according to size on agarose gels and then by shape on polyacrylamide gels. Fragments from yeast centromeres migrated faster in polyacrylamide than predicted from their base composition and size and this property was attributed to a nonrandom distribution of oligomeric A tracts that exhibited minima at 10-11 base intervals. Fragments from seven loci in 107 kb of DNA migrated anomalously slow and these fragments contained blocks of A2-6 in a 10-11 base periodicity which is indicative of bent DNA. The most pronounced bent sequences were found within yeast ARS1 and centered at 245 and 240 bp from the left and right ends of the adenovirus genome. Each sequence is approximately 150 bp away from a replication origin and the adenovirus sequences are within 50 bp of enhancers. Nuclear matrix attachment sites, which are also adjacent to enhancers, contain sequences characteristic of bent DNA. These results suggest that bent structures reside at the base of DNA loops in chromosomes.     

Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.

The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator.     

Chemical determinants of DNA bending at adenine-thymine tracts

DNA fragments having homopolymeric adenine-thymine tracts phased with the helix screw are known to be bent. According to our working model, adenine-thymine tracts adopt a polymorphic structure (H-DNA), and juxtaposition of H-DNA with B-DNA results in bending at the junction between the two structures. We incorporated different base analogues in addition to the four ordinary bases into oligonucleotides; ligated multimers of oligonucleotide duplexes were run on polyacrylamide gels. By comparison of gel mobility data for different sequences, we identified factors both necessary and irrelevant for bending, corresponding to the formation of H-DNA. The 5-methyl group on pyrimidines is not essential, and the 2-amino group on purines interferes with the formation of H-DNA, either because it provides a third H bond between the bases or because it alters water structure in the minor groove. The strong base stacking of A may be an important contributing factor to stabilization of the anomalous DNA structure responsible for bending.     

Analysis of DNA bending by transient electric birefringence

Transient electric birefringence has been used to analyze DNA bending in six restriction fragments containing 171, 174, 207, 263, 289, and 471 bp in three different low ionic strength buffers. The target fragments contain sequences corresponding to the apparent bend centers in pUC19 and Litmus 28, previously identified by the circular permutation assay (Strutz, K.; Stellwagen, N. C. Electrophoresis 1996, 17, 989-995). The target fragments migrate anomalously slowly in polyacrylamide gels and exhibit birefringence relaxation times that are shorter than those of restriction fragments of the same size, taken from nonbent regions of the same plasmids. Apparent bend angles ranging from 30 degrees to 41 degrees were calculated for the target fragments by tau-ratio method. The bend angles of four of the target fragments were independent of temperature from 4 degrees C to 20 degrees C, but decreased when the temperature was increased to 37 degrees C. The bend angles of the other two target fragments were independent of temperature over the entire range examined, 4 degrees -37 degrees C. Hence, the thermal stability of sequence-dependent bends in random-sequence DNA is variable. The bend angles of five of the six target fragments were independent of the presence or absence of Mg2+ ions in the solution, indicating most of the target fragments were stably bent or curved, rather than anisometrically flexible. Restriction fragments containing 219 and 224 bp, with sequences somewhat offset from the sequence of the 207 bp fragment, were also studied. Comparison of the tau-ratios of these overlapping fragments allowed both the bend angle and bend position to be independently determined. These methods should be useful for analyzing sequence-dependent bending in other random-sequence DNAs.     

Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor

Escherichia coli integration host factor (IHF) is a small basic protein that is required for efficient integrative recombination of bacteriophage lambda. IHF binds specifically to sequences within attP, the site in bacteriophage lambda that undergoes recombination. It has been suggested that the binding of IHF creates bends in DNA so as to help attP condense into a compact structure that is activated for recombination. In this work we show that IHF binding to either of two sites found within attP does indeed produce bending of DNA. In contrast, the other recombination protein needed for integrative recombination, Int, does not appreciably bend the DNA to which it is bound. In agreement with the proposal that IHF bending is important for creating a condensed attP, bending by IHF persists in the presence of bound Int. Our conclusions about protein-directed bends in DNA are based on the study of the electrophoretic mobility of a set of permuted DNA fragments in the presence or absence of IHF and/or Int. To facilitate this study, we have constructed a novel vector that simplifies the generation of permuted fragments. This vector should be useful in studying the bending of other DNA sequences by specific binding proteins.     

Ring closure probabilities for DNA fragments by Monte Carlo simulation

The rate of ligation of DNA molecules into circular forms depends on the ring closure probability, commonly called the j-factor, which is a sensitive measure of the extent to which thermal fluctuations contribute to bending and twisting of DNA molecules in solution. We present a theoretical treatment of the cyclization equilibria of DNA that employs a special Monte Carlo method for generating large ensembles of model DNA chains. Using this method, the chain length dependence of the j-factor was calculated for molecules. in the size range 250 to 2000 base-pairs. The Monte Carlo results are compared with recent analytical theory and experimental data. We show that a value of 475 A for the persistence length of DNA, close to values measured by a number of other methods, is in excellent agreement with the cyclization results. Preliminary applications of the Monte Carlo method to the problem of systematically bent DNA molecules are presented. The calculated j-factor is shown to be very sensitive to the amount of bending in these fragments. This fact suggests that ligase closure measurements of systematically bent DNA molecules should be a useful method for studying sequence-directed bending in DNA.     

DNA bending by small, mobile multivalent cations.

We propose a purely electrostatic mechanism by which small, mobile, multivalent cations can induce DNA bending. A multivalent cation binds at the entrance to the B-DNA major groove, between the two phosphate strands, electrostatically repelling sodium counterions from the neighboring phosphates. The unscreened phosphates on both strands are strongly attracted to the groove-bound cation. This leads to groove closure, accompanied by DNA bending toward the cationic ligand. We explicitly treat the dynamic character of the cation-DNA interaction using an adiabatic approximation, noting that DNA bending is much slower than the diffusion of nonspecifically bound, mobile cations. We make semiquantitative estimates of the free energy components of bending-electrostatic (with a sigmoidal distance-dependent dielectric function), elastic, and entropic cation localization-and find that the equilibrium state is bent B-DNA stabilized with a self-localized cation. This is a bending polaron, formation of which should be critically dependent on the strength of electrostatic interaction and the concentration of highly mobile cations available for self-localization. We predict that the resultant bend will be large (approximately 20-40 degrees), smooth (because it is spread over 6 bp), and infrequent. The stability of such a bend can be variable, from transient to highly stable (static) bending, observable with standard curvature-measuring techniques. We further predict that this bending mechanism will have an unusual sequence dependence: sequences with less binding specificity will be more bent, unless the specific binding site is in the major groove.