Cyclization Kinetics Research Articles

Bending and flexibility of methylated and unmethylated EcoRI DNA

We used cyclization kinetics experiments and Monte Carlo simulations to determine a structural model for a DNA decamer containing the EcoRI restriction site. Our findings agree well with recent crystal and NMR structures of the EcoRI dodecamer, where an overall bend of seven degrees is distributed symmetrically over the molecule. Monte Carlo simulations indicate that the sequence has a higher flexibility, assumed to be isotropic, compared to that of a “generic” DNA sequence. This model was used as a starting point for the investigation of the effect of cytosine methylation on DNA bending and flexibility. While methylation did not affect bend magnitude or direction, it resulted in a reduction in bending flexibility and under-winding of the methylated nucleotides. We demonstrate that our approach can augment the understanding of DNA structure and dynamics by adding information about the global structure and flexibility of the sequence. We also show that cyclization kinetics can be used to study the properties of modified nucleotides.

Fluorescence Study of the Coil-Globule Transition of a PEO Chain in Toluene

The cyclization kinetics of a poly(ethylene oxide) (PEO) chain (Mn = 3280; Mw/Mn = 1.05) labeled at both ends with pyrene was studied in toluene at several temperatures. Above 30 °C, the kinetics of pyrene excimer formation is well described by a two-state model. Below this temperature, a new excited species appears, which we identified as being a pyrene dimer. The appearance of the pyrene dimer was associated with a broad coil−globule transition. For lower temperatures, the kinetics of pyrene excimer and dimer formation can only be described by a three-states model. The fluorescence decay curves of the polymer in toluene were analyzed according to these models in order to obtain the rate constants. The activation energies of the cyclization and ring-opening processes were obtained from the Arrhenius plots of the rate constants. The activation energies for the cyclization processes (excimer and excited dimer formation) are close to the viscous flow activation energies. From the excimer dissociation rate c...

Study on the cyclization tendency of backbone cyclic tetrapeptides.

The cyclization kinetics of five backbone-cyclic tetrapeptides was investigated both experimentally and computationally. The aim was to both accurately measure the cyclization rates in solution and develop a method that efficiently estimates the relative cyclization tendencies computationally. Progression of the cyclization reaction was monitored directly, yielding the kinetics of changes in the amounts of the linear precursor and the products. These measurements were used to calculate the reaction rates; the results were consistent with a first-order reaction kinetics. In order to predict the cyclization rates computationally, the conformation space of the linear precursors was mapped and used to construct an approximate partition function. We assumed that the cyclization tendency was correlated with the relative probability of being found in a cyclization-prone conformation of the backbone, this probability was estimated from the partition function. The results supported this assumption and demonstrated that, within reasonable accuracy, we are able to predict the relative cyclization tendencies of the peptides measured.

Global structure and mechanical properties of a 10-bp nucleosome positioning motif.

The method of DNA cyclization kinetics reveals special properties of the TATAAACGCC sequence motif found in DNA sequences that have high affinity for core histones. Replacement of 30 bp of generic DNA by three 10-bp repeats of the motif in small cyclization constructs increases cyclization rates by two orders of magnitude. We document a 13 degrees bend in the motif and characterize the direction of curvature. The bending force constant is smaller by nearly 2-fold and there is a 35% decrease in the twist modulus, relative to generic DNA. These features are the likely source of the high affinity for bending around core histones to form nucleosomes. Our results establish a protocol for determination of the ensemble-averaged global solution structure and mechanical properties of any approximately 10-bp DNA sequence element of interest, providing information complementary to that from NMR and crystallographic structural studies.

Effects of Added CO2 on the Conformation of Pyrene End-Labeled Poly(dimethylsiloxane) Dissolved in Liquid Toluene

We report on the tail−tail cyclization and unfolding kinetics of poly(dimethylsiloxane) that is end-labeled with pyrene (Py−PDMS−Py) when it is dissolved at low concentration in liquid toluene (a good solvent) as a function of added CO2 (0−204 bar). The pyrene excimer emission provides information on the PDMS tail−tail cyclization and unfolding kinetics and the conformation of the polymer chains. Under the aforementioned conditions, the Py−PDMS−Py excimer emission is entirely intramolecular in nature; there is no evidence for any inter- or intramolecular ground-state preassociation of the pyrene residues. However, the pyrene excimer-to-monomer intensity ratio (E/M) increases by ∼5-fold as we increase the CO2 pressure from 0 to 70 bar. E/M begins to decrease gradually as the CO2 pressure is increased above 70 bar. Time-resolved fluorescence spectroscopy reveals three important points. First, the rate of Py−PDMS−Py tail−tail unfolding (kunfolding) is essentially independent of added CO2. Second, the rate th...

Designed hyperstable lac Repressor·DNA loop topologies suggest alternative loop geometries

Lac repressor (LacI) forms DNA loops which are critical for efficient operator binding and transcriptional repression. Designed DNA loops formed on three constructs with lac operators bracketing phased A-tract bends were characterized by mobility shift, footprinting, and DNA cyclization and topology. Operator dyad axes point either in or out relative to the sequence-induced curvature. Possible conformations suggested from X-ray structures of LacI and LacI·DNA include “wrapping away” (WA), “simple loop” (SL), and “wrapping toward” (WT) models. The WA loop should be preferentially stabilized by the outward operators, the SL and WT loops by the inward operators. Competition experiments demonstrated increased loop stability for all the bent constructs, with the SL/WT construct supporting hyperstable loops (t1/2 of days). This offers a general approach to stabilizing multi-protein DNA complexes on short DNA. DNA cyclization of loops gave minicircle products with altered topologies. WA constructs afforded relaxed and positive topoisomers, and cyclization kinetics indicated slow interconversion of precursors to the two topoisomers. The SL/WT construct gave a relaxed topoisomer, with a small amount of negative supercoil. These results suggest that while it is possible to force the WA loop to form (as in a model proposed from the LacI·DNA structure), the most stable loop geometry is different, probably a U-shape around an extended LacI tetramer. The topological results show how a protein-induced positive supercoil can be reconciled with LacI’s preference for binding negatively supercoiled DNA. We suggest that looping proteins can affect the assembly of subsequent proteins by controling loop topology.

Nucleosome Structural Features and Intrinsic Properties of the TATAAACGCC Repeat Sequence

Nucleosomes, the fundamental building blocks of chromatin, play an architectural role in ensuring the integrity of the genome and act as a regulator of transcription. Intrinsic properties of the underlying DNA sequence, such as flexibility and intrinsic bending, direct the formation of nucleosomes. We have earlier identified genomic nucleosome-positioning sequences with increased in vitro ability for nucleosome formation. One group of sequences bearing a 10-base pair consensus repeat sequence of TATAAACGCC had the highest reported nucleosome affinity from genomic material. Here, we report the intrinsic physical properties of this sequence and the structural details of the nucleosome it forms, as analyzed by footprinting techniques. The minor groove is buried toward the histone octamer at the AA steps and facing outwards at the CC steps. By cyclization kinetics, the overall helical repeat of the free DNA sequence was found to be 10.5 base pairs/turn. Our experiments also showed that this sequence is highly flexible, having a J-factor 25-fold higher than that of random sequence DNA. In addition, the data suggest that twist flexibility is an important determinant for translational nucleosome positioning, particularly over the dyad region.

TATA Box DNA Deformation with and without the TATA Box-binding Protein

DNA ring closure methods have been applied to TATA box DNA and its complex with the TATA box-binding protein (TBP). The J factors for cyclization (effective concentrations of one DNA end about the other) have been measured using cyclization kinetics, with and without bound TBP, for 18 DNA constructs containing the adenovirus major late promoter TATA box (TATAAAAG) separated by a variable helical phasing adapter from sequence-induced A-tract DNA bends. Six phasing lengths were used at three overall DNA lengths each. Cyclization kinetics were also measured in the absence of protein for the same set of molecules bearing a mutant TATA box (TACAAAAG). The results suggest that the TATA box DNA itself is strongly bent and anisotropically flexible, in a direction opposite to the bend induced by TBP, and that the mutant TACA box is much less bent/flexible. The bending and flexibility of the free DNA may govern the energetics of recognition of different DNA sequences by TBP, and the intrinsic bend may act to repress transcription complex assembly in the absence of TBP. The cyclization kinetics of TBP-DNA complexes in solution predict a geometry generally consistent with crystal structures, which show dramatic bending and unwinding. The novel observation of TBP-induced topoisomers suggests that this minicircle approach is able to distinguish TBP-induced unwinding from writhe (these cancel out in larger DNA), and this in turn suggests that changes in supercoiling in small topological domains can control TBP binding.

DNA-binding domains of Fos and Jun do not induce DNA curvature: an investigation with solution and gel methods.

We demonstrate the use of a DNA minicircle competition binding assay, together with DNA cyclization kinetics and gel-phasing methods, to show that the DNA-binding domains (dbd) of the heterodimeric leucine zipper protein Fos-Jun do not bend the AP-1 target site. Our DNA constructs contain an AP-1 site phased by 1-4 helical turns against an A-tract-directed bend. Competition binding experiments reveal that (dbd)Fos-Jun has a slight preference for binding to linear over circular AP-1 DNAs, independent of whether the site faces in or out on the circle. This result suggests that (dbd)Fos-Jun slightly stiffens rather than bends its DNA target site. A single A-tract bend replacing the AP-1 site is readily detected by its effect on cyclization kinetics, in contrast to the observations for Fos-Jun bound at the AP-1 locus. In contrast, comparative electrophoresis reveals that Fos-Jun-DNA complexes, in which the A-tract bend is positioned close (1-2 helical turns) to the AP-1 site, show phase-dependent variations in gel mobilities that are comparable with those observed when a single A-tract bend replaces the AP-1 site. Whereas gel mobility variations of Fos-Jun-DNA complexes decrease linearly with increasing Mg2+ contained in the gel, the solution binding preference of (dbd)Fos-Jun for linear over circular DNAs is independent of Mg2+ concentration. Hence, gel mobility variations of Fos-Jun-DNA complexes are not indicative of (dbd)Fos-Jun-induced DNA bending (upper limit 5 degrees) in the low salt conditions of gel electrophoresis. Instead, we propose that the gel anomalies depend on the steric relationship of the leucine zipper region with respect to a DNA bend.

Characterization of the ATF/CREB site and its complex with GCN4

We have studied DNA minicircles containing the ATF/CREB binding site for GCN4 by using a combination of cyclization kinetics experiments and Monte Carlo simulations. Cyclization rates were determined with and without GCN4 for DNA constructs containing the ATF/CREB site separated from a phased A-tract multimer bend by a variable length phasing adaptor. The cyclization results show that GCN4 binding does not significantly change the conformation of the ATF/CREB site, which is intrinsically slightly bent toward the major groove. Monte Carlo simulations quantitate the ATF/CREB site structure as an 8 degrees bend toward the major groove in a coordinate frame near the center of the site. The ATF/CREB site is underwound by 53 degrees relative to the related AP-1 site DNA. The effect of GCN4 binding can be modeled either as a decrease in the local flexibility, corresponding to an estimated 60% increase in the persistence length for the 10-bp binding site, or possibly as a small decrease (1 degrees) in intrinsic bend angle. Our results agree with recent electrophoretic and crystallographic studies and demonstrate that cyclization and simulation can characterize subtle changes in DNA structure and flexibility.

Looping Dynamics of Linear DNA Molecules and the Effect of DNA Curvature: A Study by Brownian Dynamics Simulation

A Brownian dynamics (BD) model described in the accompanying paper (Klenin, K., H. Merlitz, and J. Langowski. 1998. A Brownian dynamics program for the simulation of linear and circular DNA, and other wormlike chain polyelectrolytes. Biophys. J. 74:000–000) has been used for computing the end-to-end distance distribution function, the cyclization probability, and the cyclization kinetics of linear DNA fragments between 120 and 470 basepairs with optional insertion of DNA bends. Protein-mediated DNA loop formation was modeled by varying the reaction distance for cyclization between 0 and 10 nm. The low cyclization probability of DNA fragments shorter than the Kuhn length (300 bp) is enhanced by several orders of magnitude when the cyclization is mediated by a protein bridge of 10 nm diameter, and/or when the DNA is bent. From the BD trajectories, end-to-end collision frequencies were computed. Typical rates for loop formation of linear DNAs are 1.3 · 10 3 s −1 (235 bp) and 4.8 · 10 2 s −1 (470 bp), while the insertion of a 120° degree bend in the center increases this rate to 3.0 · 10 4 s −1 (235 bp) and 5.5 · 10 3 s −1 (470 bp), respectively. The duration of each encounter is between 0.05 and 0.5 μs for these DNAs. The results are discussed in the context of the interaction of transcription activator proteins.

Measurement of the DNA bend angle induced by the catabolite activator protein using Monte Carlo simulation of cyclization kinetics

A Monte Carlo simulation method for studying DNA cyclization (or ring-closure) has been extended to the case of protein-induced bending, and its application to experimental data has been demonstrated. Estimates for the geometric parameters describing the DNA bend induced by the catabolite activator protein (CAP or CRP) were obtained which correctly predict experimental DNA cyclization probabilities ( J factors), determined for a set of 11 150 to 166 bp DNA restriction fragments bearing A tracts phased against CAP binding sites. We find that simulation of out-of-phase molecules is difficult and time consuming, requiring the geometric parameters to be optimized individually rather than globally. A wedge angle model for DNA bending was found to make reasonable predictions for the free DNA. The bend angle in the CAP-DNA complex is estimated to be 85 to 90°, in agreement with estimates from gel electrophoresis and X-ray co-crystal structures. Since the DNA is found to have a pre-existing bend of 15°, the change in bend angle induced by CAP is 70 to 75°, in agreement with an estimate from topological measurements. We find evidence for slight (∼10°) unwinding by CAP. The persistence length and helical repeat of the unbound portion of the DNA are in accord with literature-cited values, but the best-fit DNA torsional modulus C is found to be 1.7(±0.2) × 10 −19 erg · cm, versus literature estimates and best-fit values for the free DNA of 2.0 × 10 −19 to 3.4 × 10 −19 erg · cm. Simulations using this low value of C predict that cyclization of molecules with out-of-phase bends proceeds via undertwisting or overtwisting of the DNA between the bends, so as to align the bends, rather than through conformations with substantial writhe. We present experiments on the topoisomers formed by cyclization with CAP which support this conclusion, and thereby rationalize the surprising result that cyclization can actually be enhanced by out-of-phase bends if the twist required to align the bends improves the torsional alignment of the ends. The relationship between the present work and previous studies on DNA bending by CAP is discussed, and recommendations are given for the efficient application of the cyclization/simulation approach to DNA bending.

Steric Effects in the Base-Catalyzed Cyclization of 1-[2-(Methoxycarbonyl)phenyl]-3-(2-substituted Phenyl)triazenes

Eleven model 1-[2-(methoxycarbonyl)phenyl]-3-(2-substituted phenyl)triazenes were synthesized and their cyclization kinetics examined in aqueous-methanolic buffer solutions (51 wt.% methanol) at various pH values. 3(2-Substituted phenyl)benzo[d][1,2,3]triazin-4(3H)-ones were identified as the cyclization products. The log kobs vs pH plot was linear with a slope of unity. Investigation of the steric and electronic effects of substituents in the ortho position revealed that substituents at the ring which is bonded to the N(3) nitrogen affect the cyclization rate through their steric effect only, while their electronic effects are statistically insignificant. This fact was explained in terms of the ring being tilted from the plane in which the remaining part of the conjugate base anion of the model substrate lies. The assumed and confirmed BAc2 mechanism involving specific base catalysis begins by deprotonation of the triazene giving rise to the conjugate base, continues with formation of a tetrahedral intermediate, and ends with elimination of the methanolate ion. Other mechanisms, such as the elimination-addition mechanism via a ketene intermediate or the mechanism involving general base catalysis, are unlikely.

Kinetics of Cyclization of Methyl S-(2,4,6-Trinitrophenyl)mercaptoacetate to 2-Methoxycarbonyl-5,7-dinitrobenzo[d]thiazol-3-oxide

The cyclization kinetics of methyl S-(2,4,6-trinitrophenyl)mercaptoacetate to 2-methoxycarbonyl-5,7-dinitrobenzo[d]thiazol-3-oxide have been studied in acetate, methoxyacetate or N-methylmorpholine buffers. In the acetate and methoxyacetate buffers, the cyclization obeys the rate equation v = [SH](k'MeO[CH3O-] + k'B[B-] + k'B,MeO[B-][CH3O-]) and goes by two reaction paths differing in the order of their reaction steps, the splitting off of the proton from C-H group being the rate-limiting step in either path. In the N-methylmorpholine buffers, increasing concentration of the base results in gradual decrease of reaction order in the base and change in the rate-limiting step of cyclization. Methyl S-(2,4-dinitrophenyl)mercaptoacetate undergoes cyclization neither in the given buffers nor in methoxide solution.

The Organic Crystallizing Agent 2-Methyl-2,4-pentanediol Reduces DNA Curvature by Means of Structural Changes in A-tracts

Contemporary predictive models for sequence-dependent DNA structure provide a good estimation of overall DNA curvature in most cases. However, the two current models differ fundamentally in their view of the origin of DNA curvature. An earlier model that associates DNA bending primarily, although not exclusively, with stretches of adenines (A-tracts) is based on results of comparative gel retardation, cyclization kinetics, hydroxyl radical cutting, and other solution measurements. It represents an intersection of wedge and junction models. More recently, a non-A-tract bending model has been proposed, built on structural results from x-ray crystallography and molecular modeling. In this view, A-tracts are proposed to be straight and rigid, whereas mixed sequence DNA is bent. Because a key premise of the non-A-tract bending model is the crystallographic observation that A-tracts are straight, we have examined the effect in solution of 2-methyl-2,4-pentanediol (MPD), an organic solvent used in crystal preparation for crystallographic DNA structure determinations. Using cyclization analysis, DNase I cutting, chemical probing, and electron microscopy on DNA oligomers with and without A-tracts, we show that the presence of MPD in solution dramatically affects A-tracts and that the effect is specific to these sequence elements. Combined with the previous observation that MPD affects gel mobility of curved sequences with A-tracts, our findings support the bent A-tract model and call for caution in the interpretation of crystallographic results on DNA structure as these are presently obtained.

Cyclization kinetics of poly(acrylonitrile)

Experimental studies of poly(acrylonitrile) (PAN) homopolymer and itaconic acid co-polymer stabilization examine the impact of polymer composition, temperature and reaction environment on the rate of cyclization. Stabilization of homopolymer PAN in nitrogen, 10.5 and 21% oxygen produces sigmoidally shaped curves when extent of cyclization is plotted as a function of reaction time. The length of the initial induction period in the sigmoidally shaped homopolymer curves decreases with increasing temperature and when oxygen is present in the reaction environment. Co-polymer cyclization curves also exhibit an induction period when reacted in nitrogen, but the period is less distinct than in the homopolymer case. The length of the induction period in the co-polymer data decreases with increasing temperature and increasing acid concentration. In the presence of oxygen, no induction period is present in any of the acidic co-polymer data. Reactions in 10.5 or 21% oxygen result in retardation of PAN homopolymer and co-polymer cyclization relative to reaction in a nitrogen environment. The retarding effect of oxygen is minimized as the acid concentration in the polymer is increased.

Synthesis of Symmetric Fluorescently Labeled Poly(ethylene glycols) Using Phosphoramidites of Pyrenebutanol and Their Characterization by MALDI Mass Spectrometry

We report the synthesis and characterization of a series of poly(ethylene oxide) [PEO] polymers in which two pyrene groups are separated by a common EO87 chain, and flanked by tails of varying EOx length. These polymers were prepared by reacting HO−EO87−OH (Mn = 3841, Mw/Mn = 1.01) with four different N,N-diisopropylphosphoramidites containing both pyrene (Py) and MeO−EOx− substituents. In this way, polymers of the form EOx−Py−EO87−Py−EOx (with MeO end groups) were obtained, with x = 2, 12, 17, and 47. These polymers are particularly useful for studying cyclization kinetics via pyrene excimer formation for two points labeled by pyrenes within the interior of a polymer chain. The polymers were characterized by NMR, size exclusion chromatography, and matrix-assisted laser desorption (MALDI) mass spectroscopy. The MALDI results were particularly informative since each of the oligomers of each of the components could be identified in each mass spectrum.

Retinoid receptors cause distortion of the retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter.

Functional retinoic acid response elements (RAREs) have been described wherein the direct repeats are separated by 1, 2 or 5 bp (termed DR1, DR2 and DR5 respectively). We have previously shown that retinoic acid receptor/retinoid X receptor (RAR/RXR) binds a DR1 RARE within the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter and is the trans-acting complex that mediates the retinoic acid (RA) response. However, the mechanism of trans-activation is unknown. The consequences of RAR/RXR binding to the PEPCK RARE were examined using a circular permutation analysis as a first step to explore the possible role of DNA conformational changes in the RA response. The RAR/RXR heterodimer produced a distortion angle of 78 degrees. The DNA distortion was shown to be at the centre of the PEPCK RARE; RA did not affect the severity of the distortion angle or the location of the distortion centre. Monomers and homodimers of RAR also distorted the DNA, but to a lesser extent than did RAR/RXR. The results of a phasing analysis demonstrated that RAR/RXR heterodimers did not induce a static DNA bend, in either the presence or the absence of RA. A cyclization kinetics assay was employed to show that RAR/RXR binding affected DNA ring closure in a phase-sensitive, RA-insensitive, manner. Taken together, these observations support the idea that RAR/RXR heterodimers distort the structure of the PEPCK RARE, at least in part, by altering DNA flexibility. The conformational change in the PEPCK RARE upon RAR/RXR binding has implications for how RAR/RXR heterodimers recognize various RARE structures.

Homodimer of p50 (NF kappa B1) does not introduce a substantial directed bend into DNA according to three different experimental assays.

Transcription factors can distort the conformation of the DNA double helix upon binding to their target sites. Previously, studies utilizing circular permutation--electrophoretic mobility shift assay suggested that the homodimer of p50 (NF kappa B1), canonical NF-kappa B (p65-p50), as well as several non-canonical NF-kappa B/Rel complexes, may induce substantial DNA bending at the binding site. Here we have applied three additional experimental approaches, helical phasing analysis, minicircle binding and cyclization kinetics, and conclude that the homodimer of p50 introduces virtually no directed bend into the consensus kappa B sequences GGGACTTTCC or GGGAATTCCC.

Detection of localized DNA flexibility.

The bending and flexibility of DNA are important in packaging, recombination and transcription. Bending decreases electrophoretic mobility in a manner depending on bend position within a fragment (circular permutation) and on the distance between bends (phasing analysis). Bending can also affect DNA ring closure (cyclization). The lack of a complete theory for the mechanism of gel retardation hampers measurement of bend magnitudes by electrophoresis, whereas cyclization is done entirely in solution and is well understood theoretically. Disagreements between bend angles estimated by the two electrophoretic assays have been ascribed to DNA flexibility. Here we test this interpretation using an internal loop as a model flexible locus. Whereas the circular permutation and helical phasing experiments are only subtly affected by the loop, DNA cyclization kinetics detects and quantifies substantial increases in torsional and bending flexibility. Furthermore, the results support a functional role for the stress of DNA bending in inducing base-pair opening.