DNA Kinking Research Articles

Nonlinear Elasticity of DNA Bending

Double stranded (ds) DNA has been used as a “molecular spring” to mechanically control the conformation of proteins, ribozymes and peptides [1]. It is thus interesting to characterize quantitatively the mechanics of DNA springs in the relevant regime of sharp bending (x<<2L<<ld, where x is the end-to-end distance (EED), 2L the contour length of the DNA, ld ∼50 nm the persistence length). This nonlinear elasticity regime is also relevant to several cell biology mechanisms, such as DNA packaging and transcription regulation.We address the problem with the stressed molecules consisting of a bent ds DNA part and a stretched ss part: essentially a system of two coupled nonlinear springs. We measured the elastic energy of these molecules with two different equilibrium methods, based on dimer formation [2, 3] and melting curve analysis [4] respectively. We deduce the bending energy of the ds part as a function of EED x. The finding is that the DNA kinks at a critical bending torque τc ∼30 pN x nm, entering a nonlinear regime where the maximum torque is constant (= τc). We derive an analytic expression for the bending energy valid in the linear and nonlinear regimes [3].The critical torque τc introduces a universal energy scale ∼12 kT in the physics of DNA bending.[1] G. Zocchi, “Controlling proteins through molecular springs”, Ann. Rev. Biophys. 38, 75-88 (2009).[2] H. Qu, C.-Y. Tseng, Y. Wang, A. J. Levine, and G. Zocchi, “The elastic energy of sharply bent nicked DNA”, Europhys. Lett. 90, 18003 (2010).[3] H. Qu and G. Zocchi, “The complete bending energy function for nicked DNA”, Europhys. Lett. 94, 18003 (2011).[4] H. Qu, Y. Wang, C.-Y. Tseng, and G. Zocchi, “Critical torque for kink formation in double stranded DNA”, accepted by Phys. Rev. X (2011).

Enantiospecific kinking of DNA by a partially intercalating metal complex

Opposite enantiomers of [Ru(phenanthroline)(3)](2+) affect the persistence length of DNA differently, a long speculated effect of helix kinking. Our molecular dynamics simulations confirm a substantial change of duplex secondary structure produced by wedge-intercalation of one but not the other enantiomer. This effect is exploited by several classes of DNA operative proteins.

In situ analysis of cisplatin binding to DNA: the effects of physiological ionic conditions

Platinum-based anti-cancer drugs form a major family of cancer chemotherapeutic agents. Cisplatin, the first member of the family, remains a potent anti-cancer drug and exhibits its clinical effect by inducing local DNA kinks and subsequently interfering with DNA metabolism. Although its mechanism is reasonably well understood, effects of intracellular ions on cisplatin activity are left to be elucidated because cisplatin binding to DNA, thus its drug efficacy, is modified by various ions. One such issue is the effect of carbonate ions: cisplatin binding to DNA is suppressed under physiological carbonate conditions. Here, we examined the role of common cellular ions (carbonate and chloride) by measuring cisplatin binding in relevant physiological buffers via a DNA micromanipulation technique. Using two orthogonal single-molecule methods, we succeeded in detecting hidden monofunctional adducts (kink-free, presumably clinically inactive form) and clearly showed that the major effect of carbonates was to form such adducts and to prevent them from converting to bifunctional adducts (kinked, clinically active). The chloride-rich environment also led to the formation of monofunctional adducts. Our approach is widely applicable to the study of the transient behaviours of various drugs and proteins that bind to DNA in different modes depending on various physical and chemical factors such as tension, torsion, ligands, and ions.

Structure determination of an intercalating ruthenium dipyridophenazine complex which kinks DNA by semiintercalation of a tetraazaphenanthrene ligand

We describe a crystal structure, at atomic resolution (1.1 Å, 100 K), of a ruthenium polypyridyl complex bound to duplex DNA, in which one ligand acts as a wedge in the minor groove, resulting in the 51° kinking of the double helix. The complex cation Λ-[Ru(1,4,5,8-tetraazaphenanthrene)(2)(dipyridophenazine)](2+) crystallizes in a 11 ratio with the oligonucleotide d(TCGGCGCCGA) in the presence of barium ions. Each complex binds to one duplex by intercalation of the dipyridophenazine ligand and also by semiintercalation of one of the orthogonal tetraazaphenanthrene ligands into a second symmetrically equivalent duplex. The result is noncovalent cross-linking and marked kinking of DNA.

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM

The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM–sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM’s N-terminal ribbon–helix–helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore.

Structural Characterization of Torsional Destabilization in DNA

Within cells, supercoiled DNA is inherently subjected to various degrees of bending and twisting, and many DNA-binding proteins are now known to be sensitive to these mechanical features of DNA. Under significant levels of bending and/or torsional stress, DNA has been shown to depart from native B-form structure and adopt a number of more energetically favorable alternate conformations. However, very little is known about the structural details of these stress-induced structures. Recently, the DNA nuclease BAL 31 was used to assay for helical destabilizations in small DNA minicircles sustaining fixed amounts of bending and torsional stresses (Nucl. Acids Res., 36(4), 2008). Here, we seek to determine if the helical destabilizations that accompany elevated levels of negative torsional stress in tightly looped minicircles produce localized structures that confer enhanced bending flexibility to the DNA structure, such as would occur in kinked DNA. Towards this end, we synthesized untwisted, 100-bp minicircles that are recognized and digested by BAL 31 and overtwisted 106-bp minicircles that are resistant to degradation by BAL 31. Using cryo-electron microscopy, we then generated three-dimensional image reconstructions of these two minicircle species. From these quantitative descriptions of the minicircle geometries, we observe no evidence of DNA kinking in either the BAL 31-sensitive, 100-bp minicircles or the BAL 31-insensitive, 106-bp minicircles. Since the torsional destabilizations that are recognized by BAL 31 were not observed to confer significant enhancements in bending flexibility, we propose that the torsional destabilizations appearing with the 100-bp minicircle relieve negative torsional stress through the formation of a left-handed dinucleotide stack with no interruption in base-stacking at the right-to-left transition, thus minimally affecting the local bending stiffness. Our observations are consistent with the formation of Z(WC)-DNA, which has been previously theorized to be the predominate form of left-handed DNA appearing in nature.

Fis-protein induces rod-like DNA bending

Fis protein can bend DNA chain with length much shorter than its persistence length. We applied single-molecule fluorescence resonance energy transfer method to probe these conformational changes. A broad distribution of end-to-end distances correlates well with the molecular dynamics simulation. The flexibility of DNA upon Fis binding is attributed to the breakages of hydrogen bonds between base pairs. DNA kinks at specific sites, instead of continuous bending. The loosening of DNA structures might have biological implications for the functions of Fis-proteins as transcription cofactors.

Functional Evolution of Bacterial Histone-Like HU Proteins

Bacterial histone-like HU proteins are critical to maintenance of the nucleoid structure. In addition, they participate in all DNA-dependent functions, including replication, repair, recombination and gene regulation. In these capacities, their function is typically architectural, inducing a specific DNA topology that promotes assembly of higher-order nucleo-protein structures. Although HU proteins are highly conserved, individual homologs have been shown to exhibit a wide range of different DNA binding specificities and affinities. The existence of such distinct specificities indicates functional evolution and predicts distinct in vivo roles. Emerging evidence suggests that HU proteins discriminate between DNA target sites based on intrinsic flexure, and that two primary features of protein binding contribute to target site selection: The extent to which protein-mediated DNA kinks are stabilized and a network of surface salt-bridges that modulate interaction between DNA flanking the kinks and the body of the protein. These features confer target site selection for a specific HU homolog, they suggest the ability of HU to induce different DNA structural deformations depending on substrate, and they explain the distinct binding properties characteristic of HU homologs. Further divergence is evidenced by the existence of HU homologs with an additional lysine-rich domain also found in eukaryotic histone H1.

Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with DNA

Cren7, a newly found chromatin protein, is highly conserved in the Crenarchaeota. The protein shows higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils in vitro and is associated with genomic DNA in vivo. Here we report the crystal structures of the Cren7 protein from Sulfolobus solfataricus in complex with two DNA sequences. Cren7 binds in the minor groove of DNA and causes a single-step sharp kink in DNA (approximately 53 degrees) through the intercalation of the hydrophobic side chain of Leu28. Loop beta 3-beta 4 of Cren7 undergoes a significant conformational change upon binding of the protein to DNA, suggesting its critical role in the stabilization of the protein-DNA complex. The roles of DNA-contacting amino acid residues in stabilizing the Cren7-DNA interaction were examined by mutational analysis. Structural comparison of Cren7-DNA complexes with Sac7d-DNA complexes reveals significant differences between the two proteins in DNA binding surface, suggesting that Cren7 and Sul7d serve distinct functions in chromosomal organization.

Dynamics of DNA-Bending in Binding Site Recognition by IHF

We present recent progress in monitoring the DNA bending dynamics in site-specific recognition by IHF, an architectural protein from E. coli that recognizes several sites on phage λ DNA, primarily by indirect readout. IHF bends the DNA at its cognate site by nearly 180° over ∼35 bp, creating two kinks in DNA stabilized by intercalation of conserved proline residues located on two β-ribbon arms that wrap around the DNA.Previous stopped-flow and laser T-jump measurements on IHF binding to its cognate H’site revealed that DNA bending in the complex occurs on ∼1-10 ms, similar to the time-scales for thermal disruption of a single base pair in B-DNA. Here we find that inserted mismatches that increase the DNA flexibility at the site of the kinks accelerate the bending rates by nearly the same factor as the corresponding increase in binding affinity. On the other hand, modifications in DNA away from the site of the kink, as well as mutations in IHF, designed to perturb specific protein-DNA contacts, leave the bending rates unchanged despite a ∼60-100-fold decrease in the binding affinity. These results support our earlier conclusion that in the transition state ensemble separating the nonspecific from the specific complex the DNA is bent/kinked, but protein-DNA interactions that stabilize the complex have not yet been made.Our measurements also reveal a rapid (∼100 microseconds) phase in the bending kinetics. In contrast to the relaxation rates for the slow phase, which are affected by modifications in the DNA at the site of the kinks, the relaxation rates for the fast phase appear to be unaffected. This rapid phase may correspond to the wrapping/unwrapping of the β-arms of the protein in a nonspecific binding mode, as IHF scans potential binding sites on genomic DNA.

Temperature dependence of circular DNA topological states

Circular double-stranded DNA has different topological states which are defined by their linking numbers. Equilibrium distribution of linking numbers can be obtained by closing a linear DNA into a circle by ligase. Using Monte Carlo simulation, we predict the temperature dependence of the linking number distribution of small circular DNAs. Our predictions are based on flexible defect excitations that resulted from local melting or unstacking of DNA base pairs. We found that the reduced bending rigidity alone can lead to measurable changes of the variance of linking number distribution of short circular DNAs. If the defect is accompanied by local unwinding, the effect becomes much more prominent. The predictions can be easily investigated in experiments, providing a new method to study the micromechanics of sharply bent DNAs and the thermal stability of specific DNA sequences. Furthermore, the predictions are directly applicable to the studies of binding of DNA-distorting proteins that can locally reduce DNA rigidity, form DNA kinks, or introduce local unwinding.

Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

We use cryo-electron microscopy (cryo-EM) to study the 3D shapes of 94-bp-long DNA minicircles and address the question of whether cyclization of such short DNA molecules necessitates the formation of sharp, localized kinks in DNA or whether the necessary bending can be redistributed and accomplished within the limits of the elastic, standard model of DNA flexibility. By comparing the shapes of covalently closed, nicked and gapped DNA minicircles, we conclude that 94-bp-long covalently closed and nicked DNA minicircles do not show sharp kinks while gapped DNA molecules, containing very flexible single-stranded regions, do show sharp kinks. We corroborate the results of cryo-EM studies by using Bal31 nuclease to probe for the existence of kinks in 94-bp-long minicircles.

Mismatch Recognition Cycle in MutS and MSH2-MSH6 from Normal Mode Analysis and Simulations

Post-replication DNA mismatch repair (MMR) is crucial in ensuring genetic fidelity in prokaryots and eukaryots. The initial step of MMR is recognition of defective DNA by MutS or its eukaryotic homologs. Binding of MutS to mismatched DNA, the subsequent initiation of repair, and eventual recovery to a mismatch scanning mode is coupled to ATPase activity in MutS. Crystal structures of MutS and the eukaryotic MSH2:MSH6 system place the ATPase domain far away from the DNA binding domains, implicating a complex allosteric mechanism.Normal mode calculations and molecular dynamics simulations of MutS and MSH2:MSH6 structures were carried out to explore the coupling between DNA binding and ATPase activity. The mode analysis reveals conserved dynamics between the bacterial and eukaryotic complexes. Individual modes correlate ATPase activity with the probing of DNA kinking that is characteristic of mismatched DNA. Furthermore, differential ATPase activity between the MutS dimer moieties as observed experimentally is coupled to release of MutS from the mismatch during repair. Based on the calculations and consistent with available experimental data, a detailed mechanistic model of the allosteric conformational changes during DNA mismatch recognition by MutS is proposed.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Single-molecule FRET measures bends and kinks in DNA

We present advances in the use of single-molecule FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on absolute distances. The resolution obtained by this single-molecule approach harbours the potential to study in detail also protein- or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not experimentally accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, we performed studies on DNA double-helices by systematically varying donor acceptor distances from 2 to 10 nm. Analysis of dye-dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic average of the FRET efficiency. We use a nonlinear conversion function based on MD simulations that allows us to include this effect in the calculation of absolute FRET distances. To obtain unique structures, we performed a quantitative statistical analysis for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. Our higher accuracy allowed the detection of sequence-dependent DNA bending by 16 degrees . For DNA with bulged adenosines, we also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32 degrees +/- 6 degrees , 56 degrees +/- 4 degrees and 73 +/- 2 degrees , respectively. Moreover, the rotation angles and shifts of the helices were calculated to describe the relative orientation of the two arms in detail.

Eukaryotic HMGB proteins as replacements for HU in E. coli repression loop formation.

DNA looping is important for gene repression and activation in Escherichia coli and is necessary for some kinds of gene regulation and recombination in eukaryotes. We are interested in sequence-nonspecific architectural DNA-binding proteins that alter the apparent flexibility of DNA by producing transient bends or kinks in DNA. The bacterial heat unstable (HU) and eukaryotic high-mobility group B (HMGB) proteins fall into this category. We have exploited a sensitive genetic assay of DNA looping in living E. coli cells to explore the extent to which HMGB proteins and derivatives can complement a DNA looping defect in E. coli lacking HU protein. Here, we show that derivatives of the yeast HMGB protein Nhp6A rescue DNA looping in E. coli lacking HU, in some cases facilitating looping to a greater extent than is observed in E. coli expressing normal levels of HU protein. Nhp6A-induced changes in the DNA length-dependence of repression efficiency suggest that Nhp6A alters DNA twist in vivo. In contrast, human HMGB2-box A derivatives did not rescue looping.

Peculiar features of kink dynamics in inhomogeneous DNA

A mathematical model was constructed for studies of the peculiar features of the dynamics of local conformational distortions (kinks) in inhomogeneous DNA. General formulas were obtained that determine the dependence of the main dynamic characteristics of kinks: size, energy, energy density and velocity, on DNA composition. Quantitative estimates were made for the characteristics of kinks activated in polynucleotide chains analogous to promoter sequences A1, A2, and A3 of bacteriophage T7.

DNA Stretching and Extreme Kinking in the Nucleosome Core

DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone–DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55° kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.

Photochemical determination of different DNA structures

The various conformations of DNA are thought to have important biological roles. Investigation of the local DNA conformational changes associated with biological events is therefore essential to an understanding of the functions of DNA. We have reported the photoreactivities of 5-halouracil in the five characteristic local DNA structures: the A, B and Z forms, protein-induced DNA kinks and the G-quadruplex form. These studies demonstrate the detailed relationships between the local DNA structures and the photochemical products of photoinduced hydrogen abstraction by the resulting uracil-5-yl radicals, and show that this photochemical method can be used to detect DNA structures. Here, we describe in detail procedures that have been developed in our laboratory for probing DNA conformations by product analysis of photoirradiated 5-halouracil-containing DNA. The protocol includes the preparation of 5-halouracil-containing DNA and the characterization of the photoproducts, and it can be completed in 2 weeks.

Structure-based Analysis of HU–DNA Binding

HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)–DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU–DNA complexes range from ∼10–14.5 kcal/mol, representing K d values in the nanomolar to low picomolar range, and a maximum stabilization of at least ∼6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.