Crystal structure of CATGGCCATG and its implications for A-tract bending models

The single-crystal x-ray analysis of orthorhombic CATGGCCATG has revealed a previously unrecognized mode of intrinsic bending in DNA. The decamer shows a smooth bend of 23 degrees over the central four base pairs, caused by preferential stacking interactions at guanine bases. The bend is produced by a roll of base pairs along their long axes, in a direction that compresses the wide major groove of the double helix. This major-groove-compressing bend at GGC, plus the abundant crystallographic evidence that runs of successive adenine bases (A-tracts) are straight and unbent, requires rethinking of the models most commonly invoked to explain A-tract bending. A decade of excellent experimental work involving gel migration experiments, cyclization kinetics, and nucleosome phasing has clearly established that introduction of short A-tracts into a general DNA sequence in synchrony with the natural repeat of the helix leads to bending. But it does not logically and inevitably follow that the actual bending is to be found within these introduced A-tracts or even at junctions with general-sequence B-DNA.     

Distribution of striatin, a newly identified calmodulin-binding protein in the rat brain: an in situ hybridization and immunocytochemical study

The worm-like chain model has often been employed to describe the average conformation of long, intrinsically straight polymer molecules, including DNA. The present study extends the applicability of the worm-like chain model to polymers containing bends or sections of different flexibility. Several cases have been explicitly considered: (i) polymers with a single bend; (ii) polymers with multiple coplanar bends; (iii) polymers with two non-coplanar bends; and (iv) polymers comprised of sections with different persistence lengths. Expressions describing the average conformation of such polymers in terms of the mean-square end-to-end distance have been derived for each case. For cases (i) and (iv), expressions for the projection of the end-to-end vector onto the initial orientation of the chain are presented. The expressions derived here have been used to investigate DNA molecules with sequence-induced bending (A-tracts). Mean-square end-to-end distance values determined from a large number of A-tract containing DNA molecules visualized by scanning force microscopy resulted in an average bend angle of 13.5 degrees per A-tract. A similar study was performed to characterize the flexibility of double-strandedDNA molecules containing a single-stranded region. Analysis of their mean-square end-to-end distance yielded a persistence length of 1.3 nm for single-stranded DNA.     

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.     

Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT)

The crystal structure of EcoRV endonuclease has been determined at 2. 1 A resolution complexed to two five-base-pair DNA duplexes each containing the cognate recognition half-site. The highly localized 50 degrees bend into the major groove seen at the center TA-step of the continuous GATATC site is preserved in this discontinuous DNA complex lacking the scissile phosphates. Thus, this crystal structure provides evidence that covalent constraints associated with a continuous target site are not essential to enzyme-induced DNA bending, even when these constraints are removed directly at the locus of the bend. The scissile phosphates are also absent in the crystal structure of EcoRV bound to the non-specific site TCGCGA, which shows a straight B-like conformation. We conclude that DNA bending by EcoRV is governed only by the sequence and is not influenced by the continuity of the phosphodiester backbone. Together with other data showing that cleavable non-cognate sites are bent, these results indicate that EcoRV bends non-cognate sites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar. Structural and thermodynamic considerations suggest that the sequence-dependent energy cost of DNA bending is likely to play an important role in determining the specificity of EcoRV. This differential cost is manifested at the binding step for bent non-cognate sequences and at the catalytic step for unbent non-specific sequences.     

GC rich DNA oligonucleotides with narrow minor groove width

Investigation of the width of the minor groove using 500 MHz NMR spectroscopy in three closely related 11-mer B-DNA duplexes shows that the minor groove is narrow in a GC rich oligonucleotide, and that a narrow minor groove is not something endemic to DNAs with persistent repetitions of adenine nucleotides (A-tract DNA). The width of the groove is dictated by local sequence contexts and independent of neighboring A-tract DNA.     

Comparison of DNA bending by Fos-Jun and phased A tracts by multifactorial phasing analysis

Studies of DNA bending by Fos and Jun using different methods have yielded contradictory results. Whereas gel electrophoretic phasing analysis indicates that Fos and Jun bend DNA, results obtained through X-ray crystallography and ligase-catalyzed cyclization suggest that they do not. To test the assumptions underlying phasing analysis and to examine DNA bending by Fos and Jun, a multifactorial phasing analysis approach based on the distinct electrophoretic mobilities of DNA fragments of diverse shapes was developed. In this approach, the spacing between the bends, the length of sequences flanking the bends, and the acrylamide concentration in the gel are varied. Two closely spaced intrinsic bends with long flanking sequences had the same effect on electrophoretic mobility as a single bend corresponding to the sum of the bends when they were arranged in phase, and the difference between the bends when they were arranged out of phase. Based on the phase-dependent electrophoretic mobility variation of fragments containing intrinsic DNA bends of different magnitudes, three criteria for determination whether the phase-dependent mobility variation of protein-DNA complexes is caused by DNA bending were adopted. Complexes formed by the bZIP domains of Fos and Jun fulfilled each of these criteria. First, the electrophoretic mobility variation induced by Fos and Jun was proportional to that caused by an intrinsic bend over a broad range of acrylamide concentrations. Second, the mobility difference between fragments containing in phase and out of phase bends was reduced by an increase in the separation between the bends. The separation between the bends had the same effect on the electrophoretic mobility variation caused by Fos and Jun as well as intrinsic bends on long DNA fragments at low acrylamide concentrations. Third, on short DNA fragments analyzed at high acrylamide concentrations, two intrinsic bends separated by long spacers caused a larger decrease in electrophoretic mobility when they were out of phase than when they were in phase. This reversal of the phase dependence of the electrophoretic mobility variation was also observed for complexes formed by truncated Fos and Jun. Thus, the phase-dependent mobility variation of Fos and Jun complexes is due to DNA bending.     

DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.     

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 degrees, 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 degrees, the change in bend angle induced by CAP is 70 to 75 degrees, in a agreement with an estimate from topological measurements. We find evidence for slight (approximately 10 degrees) 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) x 10(-19) erg. cm, versus literature estimates and best-fit values for the free DNA of 2.0 x 10(-19) to 3.4 x 10(-19) erg.com. 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.     

Structural analysis of DNA bending induced by tethered triple helix forming oligonucleotides

In order to monitor DNA flexibility, we have recently reported the design of an artificial DNA bending system consisting of two triple helix forming oligonucleotides (TFOs) connected by a flexible linker [Akiyama, T., & Hogan, M. E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12122-12127], which spans a single turn of DNA helix. Those data suggested that up to 60 degrees of bending deformation could be induced with an expenditure of energy which is much smaller than predicted from bulk flexibility parameters. In this report, the detailed structure of the bend has been investigated by three different methods: circular permutation analysis, phasing analysis, and ring closure. Circular permutation and phasing analysis suggest that the magnitude of the bend is dependent on linker length. The apparent location of the bend was estimated from circular permutation analysis to be at the duplex region intervening the two sites of triple helix formation. The electrophoretic mobility of the bent complex appears to vary with the sequence of the intervening duplex region of the binding site complex, in the order of AT-rich > random > or = GC-rich sequence. Detailed fitting of the phasing data has shown that bending is not accompanied by significant twisting deformation. Ring closure analysis with T4 DNA ligase has confirmed the general magnitude of the TFO-induced bend and has additionally suggested that formation of the simple linear antiparallel triple helix does not enhance DNA flexibility.     

Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+

We have used the method of Zinkel and Crothers (Zinkel, S.S., and Crothers, D.M. (1990) Biopolymers 29, 29-38) to determine the degree of bending induced by the binding of the restriction endonuclease EcoRV to its recognition sequence (-GATATC-). A set of four calibration DNA fragments was constructed that contained zero, two, four, or six phased A-tracts in their centers and an EcoRV site at the 5'-end to account for the electrophoretic influence of the bound protein. The mobilities of these calibration molecules complexed with EcoRV were compared to that of a test DNA containing a central EcoRV site also complexed with EcoRV. The EcoRV-induced bend angle was found to be 44 degrees +/- 4 degrees. These experiments were performed with a catalytically inactive EcoRV mutant that still binds DNA specifically in the presence of Mg2+. In the absence of Mg2+, which is necessary for specific binding, there is no difference in the mobilities of the fragments with a peripheral or a central EcoRV site complexed with EcoRV, indicating that nonspecific binding on average does not lead to measurable DNA bending.     

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting

Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90 degrees. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein--DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.     

Transient disruptions of axonemal structure and microtubule sliding during bend propagation by Ciona sperm flagella

Demembranated sperm flagella of Ciona were reactivated at increased salt concentrations (0.45 to 0.5 M K acetate). In addition to a decrease in amplitude of propagated bends, some flagella switch between "stable" and "transient" bending cycles. In the transient bending cycles, there is increased intermicrotubule sliding, in the direction that forms a new principal bend at the base of the flagellum, during the first half of a bending cycle. The magnitude of this increased sliding may be as much as 1 radian, or 0.06 micron between adjacent doublet microtubules. Most transient bending patterns also show a characteristic disruption of axonemal structure, involving separation between strands of microtubule doublets over a distance of up to 5 microns, occurring within a principal bend, typically about 16 microns from the base of the flagellum. The disruptions usually disappear after the principal bend propagates beyond the region of the disruption. Formation of these disruptions requires additional sliding, in the direction that would form a principal bend at the base of the flagellum, of up to about 0.3 micron. Formation of these disruptions may be explained by weakening of structural interactions by increased salt concentration and transverse forces, proportional to curvature and transmitted force, that will tend to separate doublets in a bend. These observations indicate that an actively beating flagellum possesses active sliding capability that is activated but not expressed during normal bend initiation and propagation. The initiation and propagation of flagellar bends may not be explicable solely in terms of local activation and inactivation of dynein-driven sliding.     

DNA bending: the prevalence of kinkiness and the virtues of normality

DNA bending in 86 complexes with sequence-specific proteins has been examined using normal vector plots, matrices of normal vector angles between all base pairs in the helix, and one-digit roll/slide/twist tables. FREEHELIX, a new program especially designed to analyze severely bent and kinked duplexes, generates the foregoing quantities plus local roll, tilt, twist, slide, shift and rise parameters that are completely free of any assumptions about an overall helix axis. In nearly every case, bending results from positive roll at pyrimidine-purine base pair steps: C-A (= T-G), T-A, or less frequently C-G, in a direction that compresses the major groove. Normal vector plots reveal three well-defined types of bending among the 86 examples: (i) localized kinks produced by positive roll at one or two discrete base pairs steps, (ii) three-dimensional writhe resulting from positive roll at a series of adjacent base pairs steps, or (iii) continuous curvature produced by alternations of positive and negative roll every 5 bp, with side-to-side zig-zag roll at intermediate position. In no case is tilt a significant component of the bending process. In sequences with two localized kinks, such as CAP and IHF, the dihedral angle formed by the three helix segments is a linear function of the number of base pair steps between kinks: dihedral angle = 36 degrees x kink separation. Twenty-eight of the 86 examples can be described as major bends, and significant elements in the recognition of a given base sequence by protein. But even the minor bends play a role in fine-tuning protein/DNA interactions. Sequence-dependent helix deformability is an important component of protein/DNA recognition, alongside the more generally recognized patterns of hydrogen bonding. The combination of FREEHELIX, normal vector plots, full vector angle matrices, and one-digit roll/slide/twist tables affords a rapid and convenient method for assessing bending in DNA.     

Microbial invasion of the amniotic cavity with Ureaplasma urealyticum is associated with a robust host response in fetal, amniotic, and maternal compartments

Repression of the divergent nagE - B operons requires NagC binding to two operators which overlap the nagE and nagB promoters, resulting in formation of a DNA loop. Binding of the cAMP/CAP activator to its site, adjacent to the nagE operator, stabilizes the DNA loop in vitro. The DNA of the nagE-B intergenic region is intrinsically bent, with the bend centred on the CAP site. We show that displacement of the CAP site by 6 bp results in complete derepression of the two operons. This derepression is observed even in the absence of cAMP/CAP binding and despite the fact that the two NagC operators are still in phase, demonstrating that the inherently bent structure of the DNA loop is important for repression. Since no interaction between NagC and CAP has been detected, we propose that the role of CAP in the repression loop is architectural, stabilizing the intrinsic bend. The cAMP/CAP complex is necessary for activation of the nagE-B promoters. In this case protein-protein contacts between CAP and RNA polymerase are necessary for full activation, but at least a part of the activation is likely due to an effect of CAP binding altering DNA structure.