Central Bending Research Articles

Electric birefringence of kilobase-sized DNA molecules

Transient electric birefringence has been used to characterize the rotational diffusion of linear, circularly permuted pBR322 and SV40 DNA molecules. The birefringence relaxation times vary with the site of linearization, suggesting that the circularly permuted DNAs have different conformations in solution. The longest relaxation times are observed for DNA sequence isomers linearized at the major bend centers identified by gel electrophoresis 1 1 N.C. Stellwagen, Electrophoresis, 15 (1995) 691. . SV40 sequence isomers linearized at other locations have faster, but approximately equal, terminal relaxation times, suggesting that their free solution conformations are relatively independent of the location of the bend center within the sequence. By contrast, the terminal relaxation times of the various pBR322 sequence isomers vary approximately in accord with their electrophoretic mobilities in large-pore polyacrylamide gels, suggesting that the different mobilities may reflect real conformational differences between the sequence isomers.

Single Base-pair Precision and Structural Rigidity in a Small IHF-induced DNA Loop

The prokaryotic integration host factor (IHF) is a DNA-binding protein that binds to specific DNA sites as a heterodimer. Genetic and mutational analyses have previously identified asymmetric protein-DNA contacts by the individual subunits. By exploiting the unique sequence and positional context of one IHF binding site, H′ in Lambda attachment sites ( attsites), we have identified a symmetry element of binding and have localized the functional bend center to the center of this symmetry. A shift of the H′ bend center by a single base-pair to the right or to the left within the very tight loop formed with Lambda integrase (int) and IHF in att-site “intrasomes” severely reduces recombination. This suggests that a precise, but wrongly positioned, DNA bend within a loop constant length negatively influences the juxtaposition or “phasing” of the core-type and arm-type Int binding sites by differentially affecting the length of each leg of the loop. Furthermore, ten base-pair insertions within this loop that should not interfere with correct helical phasing are sensed in a position-dependent manner. Distal insertions abolish recombination, whereas proximal or double insertions (in both legs of the loop) are well tolerated.

High Angle and Ligand-induced Low Angle DNA Bends Incited by OccR Lie in the Same Plane with OccR Bound to the Interior Angle

The OccR protein ofAgrobacterium tumefaciensactivates transcription of theoccQoperon in response to octopine. Octopine shortens the DNase I footprint of OccR and relaxes an OccR-incited DNA bend. In this study we used hydroxyl radical footprinting to show that OccR contacts only one face of the operator. This contact spans 50 nucleotide pairs in the absence of octopine, and 39 nucleotide pairs in the presence of octopine. Octopine enhanced protection of the central region of the footprint. OccR blocked intercalation by methidiumpropyl EDTA along most of the operator, but did not block intercalation at the bend center. OccR protected DNA against both reagents far more completely at the promoter-distal half of the operator than at the promoter-promixal half. Experiments to alter the phasing between the operator bend and an adjacent sequence-directed bend indicated that the high angle DNA bend in the absence of octopine, and the low angle bend in the presence of octopine, lie in the same plane. Operator DNA was bent toward the helical face that OccR protected from hydroxyl radicals, indicating that OccR occupies the interior angle of this bend.

The Role of DNA Bending in Flp-mediated Site-specific Recombination

The Flp recombinase of the 2 μm plasmid of Saccharomyces cerevisiaebinds to a recognition target site, induces DNA bending and catalyses DNA cleavage and strand exchange to bring about recombination. The minimal Flp recognition target site contains two Flp binding sequences flanking an 8 bp core region; binding of Flp results in the formation of two Flp:DNA complexes (complexes I and II). Binding of a Flp monomer to a single symmetry element generates a DNA bend of about 60 ° (a type I bend), whereas binding of two Flp monomers to the FRT site generates a DNA bend of>144 ° (a type II bend). We have used circular permutation analysis to locate the centre of the type I and type II DNA bends induced by Flp, and the Flp peptides P27 (27 kDa; amino acid residues 124 to 346) and P32 (32 kDa; amino acid residues 124 to 423). The location of the centre of the type I bend depends upon whether the substrate contains one or two Flp binding elements. When the substrate contains one symmetry element, the centre of the type I bend induced by Flp is located at the core-distal end of the belement. However, it is located at the core-proximal end of the belement when the substrate contains two Flp-binding elements. The P27 and P32 peptides, which lack the NH 2-terminal 13 kDa region of Flp, do not show this behavior. We deduce that the 13 kDa region of Flp is critical for the positioning of the type I bend centre on a minimal Flp recognition site. We propose a model in which a single molecule of Flp interactions with two symmetry elements to account for these results. The centre of the type II bend induced by Flp is in the middle of the core region. We used ligation-defective Flp proteins to determine the location of the type II bend centres in complexes where either the top or bottom strand was cleaved. The bend centres of such complexes depend upon which strand is cleaved. We propose a model which associates the position of Flp-induced type II bends with a defined order of strand exchanges in the recombination reaction.

Bent DNA in the Large Intergenic Region of Wheat Dwarf Geminivirus

Wheat dwarf virus (WDV) is a member of the geminivirus group, unique plant DNA viruses which replicate exclusively via dsDNA replication intermediates. The large intergenic region (LIR), a nontranscribed regulatory region, contains an inverted repeat with the potential to form a stem-loop structure in which the initiation site for WDV (+)strand (virion-sense) DNA replication has been mapped. In this work, we have studied by two-dimensional electrophoresis and by electron microscopy the DNA structure of the WDV LIR and we have identified a DNA sequence which confers a static DNA curvature. This is the first report of the occurrence of DNA bending in the regulatory region of a geminivirus genome. The bending locus, defined by an 80-bp cluster of A(T) tracts, is located downstream from the potential stem-loop. The A(T) tract proximal to the stem-loop structure is separated by 14 bp from the inverted repeat, and deletion of distal A(T) tracts abolished the curvature of the new WDV ΔLIR. The bending center maps ∼80 bp downstream from the putative initiation site for (+)strand DNA replication and ∼70 bp upstream from the TATA box for virion-sense transcription. The possible implications of the DNA bending locus as a regulatory element of WDV DNA replication and/or virion-sense transcription are discussed.

An intrinsically bent region upstream of the transcription start site of the rRNA genes of Arabidopsis thaliana interacts with an HMG-related protein.

Intrinsically bent DNA structures are thought to play crucial roles as functionally important modules in promoter/enhancer elements. Here we show the existence of a DNA bending centre within the intergenic region of the tandemly arranged rRNA genes of Arabidopsis thaliana. By use of a circular permutation assay, this bending centre was localized to position -284 to -256 upstream of the transcription start site. A 143 bp fragment containing the bent region was shown to interact with several nuclear proteins. Evidence is presented that one of these proteins is related to the high-mobility-group (HMG) proteins, a group thought to be involved in transcription and replication processes.

The nonconserved hinge region and distinct amino-terminal domains of the ROR alpha orphan nuclear receptor isoforms are required for proper DNA bending and ROR alpha-DNA interactions.

ROR alpha 1 and ROR alpha 2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3' nuclear receptor core half-site AGGTCA preceded by a 5' AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR alpha 1 and ROR alpha 2 to the RORE induces a large DNA bend of approximately 130 degrees which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3' AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR alpha is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR alpha protein does not alter the DNA bend angle but shifts the DNA bend center 5' relative to the bend induced by intact ROR alpha. Methylation interference studies using the NTD-deleted ROR alpha 1 mutant indicate that some DNA contacts in the 5' AT-rich half of the RORE are also shifted 5', while those in the 3' AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR alpha NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR alpha DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.

DNA bending by Saccharomyces cerevisiae ABF1 and its proteolytic fragments.

The ABF1 protein of the yeast Saccharomyces cerevisiae has been found to bend the DNA containing the target site for DNA binding. A bend angle of about 120 degrees was measured and the bend center was 7 base pairs to the 5' end of the ABF1 consensus-containing sequence. Phasing analysis showed that intact ABF1 bends the DNA towards the minor groove. We have subjected ABF1 to partial proteolysis and have found that proteolytic fragments were able to bind to the DNA-binding site and induce partial bends in the DNA. Interestingly, the locations of the bend centers, the bend angles, and the plane of the bends induced by the proteolytic peptides of ABF1 were different from those of the intact protein. We present a model for the mechanism of bending of DNA by ABF1.

The General Model of Free Vibrations of Mistuned Bladed Discs, Part II: Numerical Results

This paper is divided into two parts. In Part I the equations of motion describing the free vibration of bladed disc assemblies were formulated. The numerical results for the tuned and mistuned bladed discs for blades of complex geometry are the subject of Part II. Answers to the question "Which of the blades in the mistuned system are most likely to experience the largest vibration amplitude?" are presented. The best and worst distributions of the detuned blades from the stress point of view are shown. The influence of the stagger angle, the cross-sectional area and the twist angle of the blades on the natural frequencies, stress distributions of the blades around the disc and the position of the maximal stress along the blade length of rotating and non-rotating tuned and mistuned bladed discs is presented. The accuracy of numerical calculations of the blades' stresses is dependent on the inclusion in the blade model of the bending centre. This problem is also considered.

Sequence determinants of DNA bending in the ilvlH promoter and regulatory region of Escherichia coli.

Previous studies have shown that the promoter/regulatory region of the ilvlH operon displays intrinsic curvature, with the bend center located at position -120 relative to the transcription start site. In this report, a 57 bp sequence spanning the bend center was mutagenized in vitro in order to study the relationship between nucleotide sequence and curvature measured by electrophoresis. The strategy used for analyzing the results consisted of determining the strengths of the relationships between electrophoretic anomaly and predicted curvature calculated by computer programs that differ in wedge angle composition. The results revealed that programs which assume that bending occurs only at AA/TT display good predictive value, with correlation coefficients between electrophoretic anomaly and predicted curvature as high as 0.93. In contrast, a program which assumes that bending occurs at all 16 dinucleotide steps exhibited lower predictive value, while there were no significant relationships between the experimental data and curvature calculated by a program that was based on all non-AA/TT wedge values. These results show that the complete wedge model which incorporates values for all dinucleotide steps does not adequately describe the electrophoretic data in this report.

Localization of the intrinsically bent DNA region upstream of the E.coli rrnB P1 promoter.

DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.

The SRY high-mobility-group box recognizes DNA by partial intercalation in the minor groove: a topological mechanism of sequence specificity.

SRY, a putative transcription factor encoded by the sex-determining region of the human Y chromosome, regulates a genetic switch in male development. Impairment of this switch leads to intersex abnormalities of the newborn and is observed in association with mutations in the SRY DNA-binding domain [the high-mobility-group (HMG) box]. Here we show that the SRY HMG box exhibits a novel mechanism of DNA recognition: partial intercalation of a nonpolar side chain in the DNA minor groove. Base stacking (but not base pairing) is interrupted at the site of insertion. Sequence specificity reflects topological requirements of partial intercalation rather than direct readout of base-specific functional groups. Our results predict that the SRY HMG box inserts an alpha-helix into a widened minor groove at the center of a sharp DNA bend. A similar mechanism may underlie binding of SRY and homologous HMG proteins to four-way junctions (Holliday intermediates) and other noncanonical DNA structures.

DNA insertions as a component of the evolution of unique satellite DNA families in two genera of parasitoid wasps: Diadromus and Eupelmus (Hymenoptera).

In the two parasitoid wasps, Diadromus collaris and Eupelmus orientalis, the satellite DNAs were each found to consist wholly or largely of a single family (5%-7% of the genome). Several clones of each family were obtained and sequenced. The repeat unit in each species is characterized by both the repetition of a basic motif and the presence of an inserted sequence. Sequence comparisons with satellite DNA from D. pulchellus and E. vuilleti provide plausible scenarios for the evolution of the satellite DNA in each genus. Palindromes and A-rich tracts in each consensus sequence suggest the formation, in vivo, of hairpin structures and bend centers that may play a role in heterochromatin condensation in insects. The insertions in the repeat units of each species also contain these structural features, suggesting that maintenance of these insertions requires constraints similar to those pertaining to the rest of the satellite-DNA unit.

Thyroid hormone receptor-induced bending of specific DNA sequences is modified by an accessory factor.

Transcriptional regulation by thyroid and steroid hormone receptors requires their recognition and binding of specific DNA sequences. However, little is known about the mechanisms whereby DNA bound receptors regulate transcription. In the present study, we examined the effects of thyroid hormone receptor (TR) binding on DNA conformation using various TR recognition sites contained within sets of circularly permuted flanking sequences. We show that under conditions where TR binds predominantly as monomer, the conformation of a number of binding sites is changed in a manner consistent with receptor induced bending. Despite similar affinities for receptor binding, not all binding sites tested showed evidence for receptor-induced bending. Notably, the conformation of a sequence from the frog vitellogenin 2 gene, which confers a positive transcriptional response when bound by estrogen receptor (ER), but a negative response when bound by TR, appeared to be unaffected by binding of either TR or ER. The observations suggest that the ability of the receptor to alter DNA architecture is strongly dependent on sequence characteristics other than those required for receptor binding. While both partly purified TR from rat liver and TR translated in vitro were able to induce DNA bending, the bend centers and bend angles produced by these different sources of receptor differed. However, addition of a receptor-depleted fraction from the rat liver TR preparation to in vitro translated receptor stimulated TR binding and appeared to form heterodimers with TR. This resulted in changes in both bend centers and bend angles to resemble more closely those produced by native receptor. Together, these results suggest that receptor-induced DNA bending may be specific to TRs and that the position and degree of bending is further modulated by the formation of heterodimers between TRs and accessory protein(s).

Swirling turbulent flow through a curved pipe

An experimental study of swirling turbulent flow through a curved bend and its downstream tangent has been carried out. This study reports on the recovery from swirl and bend curvature and relies on measurements obtained in the downstream tangent and data reported in Part 1 to assess the recovery. Unlike the nonswirling flow case, the present measurements show that the cross-stream secondary flow is dominated by the decay of the solid-body rotation and the total wall shear stress measured at the inner and outer bend (furthest away from the bend center of curvature) is approximately equal. The shear distribution is fairly uniform, even at 1 D downstream of the bend exit. At 49D downstream of the bend exit, the mean axial velocity has recovered to its measured profile at 18D upstream of the bend entrance. Furthermore, the mean tangential velocity is close to zero everywhere and the turbulent shear and normal stresses take another 15D to approximately approach their stationary straight pipe values. Therefore, complete flow recovery from swirl and bend curvature takes a total length of about 85D from the bend entrance. This compares with a recovery length of about 78D for bend curvature alone. The recovery length is substantially shorter than that measured previously in swirling flow through straight pipes and is a consequence of the angular momentum decreasing by approximately 74% across the curved bend. Consequently, the effect of bend curvature is to accelerate swirl decay in a pipe flow.

Cpf1 protein induced bending of yeast centromere DNA element I.

The centromere complex is a multicomponent structure essential for faithful chromosome transmission. Here we show that the S. cerevisiae centromere protein Cpf1 bends centromere DNA element I (CDEI) with the bend angle ranging from 66 degrees to 71 degrees. CDEI DNA sequences that carry point mutations which lead to reduced Cpf1 binding affinity and in vivo centromere activity are still able to show bending. The Cpf1 induced bend is directed towards the major groove with the bend centre located in CDEI. An intrinsic bend cannot replace the Cpf1 induced DNA bend for in vivo centromere function. An in vivo phasing experiment suggests that both the distance and the correct spatial arrangement of the CDEI/Cpf1 complex to CDEII and CDEIII are important for optimal centromere function.

Zinc induces a bend within the transcription factor IIIA-binding region of the 5 S RNA gene

Binding of Zn 2+ to the 5 S RNA gene sequence of Xenopus borealis results in strong bending of the DNA, as inferred from transient electric birefringence data. The effect is specific for Zn 2+; several other divalent ions are not able to induce a bend of a similar magnitude. Using five different fragments that span the binding sequence, we are able to estimate a bend magnitude of at least 55 ° centered at base-pair +65 within the gene. This places the bend within the binding domain of the gene-regulatory protein transcription factor (TF) IIIA. Recent evidence has shown that the protein-DNA complex is also bent. Although our data do not allow us directly to link the two bends, our results suggest that TFIIIA could form a folded structure by stabilizing the same bent conformation that is induced by binding of Zn 2+. The chemistry of Zn 2+ binding to DNA, and the sequence around the bend center, suggest that the bend is most probably caused by joint co-ordination of Zn 2+ to the N-7 groups of stacked purine residues.

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.

Binding of the arginine repressor of Escherichia coli K12 to its operator sites

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of l-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70° and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressers studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.

FIS-induced bending of a region upstream of the promoter activates transcription of the E coli thrU (tufB) operon

The upstream activator sequence (UAS) of the thrU (tufB) operon, which is the target of the trans-activating protein FIS, has a bent structure. Here we show that the center of bending lies around position — 95, between the two FIS-binding regions. Studies with fis+ and fis− cells show that FIS-induced bending of the UAS plays a major role in the trans-activation of the thrU (tufB) operon. This has been concluded from the finding that insertions of small DNA segments, comprising less than one or two complete helix turns, in the junction of the UAS and the RNA polymerase-binding site reduce transcription significantly. Partial restoration of transcriptional actioity occurs when one or more full helix turns are inserted. These data are in line with but do not prove that a direct interaction between FIS and RNA polymerase in invilved in trans-activation. A role of bending per se resulting from FIS/DNA interaction cannot be excluded.