DNA Kinking Research Articles

Molecular Origin of DNA Kinking by Transcription Factors.

Binding of transcription factor (TF) proteins with DNA may cause severe kinks in the latter. Here, we investigate the molecular origin of the DNA kinks observed in the TF-DNA complexes using small molecule intercalation pathway, crystallographic analysis, and free energy calculations involving four different transcription factor (TF) protein-DNA complexes. We find that although protein binding may bend the DNA, bending alone is not sufficient to kink the DNA. We show that partial, not complete, intercalation is required to form the kink at a particular place in the DNA. It turns out that while amino acid alone can induce the desired kink through partial intercalation, protein provides thermodynamic stabilization of the kinked state in TF-DNA complexes.

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

High-resolution mapping of architectural DNA binding protein facilitation of a DNA repression loop in Escherichia coli

Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

The Cruciform DNA Mobility Shift Assay: A Tool to Study Proteins That Recognize Bent DNA.

So-called architectural DNA-binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA-binding characteristics difficult if not impossible in a standard electrophoretic mobility shift assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity. A synthetic cruciform DNA structure will often provide an ideal binding site for such proteins, allowing their affinities for both bent and linear DNAs to be directly and simply determined by a modified form of EMSA.

Controlled dehydration of a ruthenium complex-DNA crystal induces reversible DNA kinking.

Hydration-dependent DNA deformation has been known since Rosalind Franklin recognized that the relative humidity of the sample had to be maintained to observe a single conformation in DNA fiber diffraction. We now report for the first time the crystal structure, at the atomic level, of a dehydrated form of a DNA duplex and demonstrate the reversible interconversion to the hydrated form at room temperature. This system, containing d(TCGGCGCCGA) in the presence of Λ-[Ru(TAP)2(dppz)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene, dppz = dipyrido[3,2-a:2',3'-c]phenazine), undergoes a partial transition from an A/B hybrid to the A-DNA conformation, at 84-79% relative humidity. This is accompanied by an increase in kink at the central step from 22° to 51°, with a large movement of the terminal bases forming the intercalation site. This transition is reversible on rehydration. Seven data sets, collected from one crystal at room temperature, show the consequences of dehydration at near-atomic resolution. This result highlights that crystals, traditionally thought of as static systems, are still dynamic and therefore can be the subject of further experimentation.

Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active “sliding clamp” conformation on Tg-DNA is problematic.

Minor groove geometry determines DNA exclusion from nucleosomes (929.1)

Like all eukaryotes, the meters of DNA in each of our cells must be compacted many thousands of times to fit inside the nucleus. The first level of compaction, spooling of DNA onto a nucleosome, involves the tight wrapping of DNA around eight histone proteins. Sharply bending DNA to wrap it into nucleosomes comes at an energetic cost related to the stiffness of the DNA. Nucleosomes prefer to form on DNA sequences that have a 10 bp periodicity between AA/AT/TA steps, with CC/CG/GC steps exactly out of phase. The sites of the AA/AT/TA and CC/CG/GC steps exactly correspond to locations of the highest DNA kinking, suggesting that these sequences represent anisotropically bendable sequences. Yet, inclusion of multiple AA steps, in the form of a poly(dA:dT) elements, results in exclusion of the DNA from nucleosomes in vivo. We seek to understand what unique chemical features of these poly‐dA elements are necessary for their exclusion from nucleosomes. Poly‐dA elements are known to adopt a unique structure; characterized crystallographically as a narrowed minor groove and by CD as a double peaked spectrum. Using CD spectroscopy and non‐natural nucleotides, we show that correct minor groove geometry at the base pair level is necessary for formation of the unique poly‐dA structure. Using in vitro competitive nucleosome reconstitution assays, we have further shown that non‐natural DNA sequences that adopt the unique poly‐dA structure are excluded from nucleosomes. Since poly‐dA like sequences are excluded from nucleosomes, this suggests that the unique structure formed by these sequences is energetically stiffer than generic sequence DNA.

Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SK(bi) ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SK(uni) < 0.5. This result is counterintuitive because there are at least twice as many ionic protein-DNA contacts in the fully wrapped complex than in the weakly bent intermediate. The following picture emerges from this analysis: in the bimolecular step, the observed [KCl]-dependence is consistent with the number of DNA counterions expected to be released when IHF binds nonspecifically to DNA whereas in the unimolecular reorganization step, the weak [KCl]-dependence suggests that two effects cancel one another. On one hand, formation of additional protein-DNA contacts in the fully wrapped complex releases bound counterions into bulk solution, which is entropically favored by decreasing [salt]. On the other hand, formation of the fully wrapped complex also releases tightly bound water molecules, which is osmotically favored by increasing [salt]. More generally, our global analysis strategy is applicable to other protein-DNA complexes, and opens up the possibility of measuring DNA bending rates in complexes where the unimolecular and bimolecular steps are not easily separable.

Euler buckling and nonlinear kinking of double-stranded DNA

The bending stiffness of double-stranded DNA (dsDNA) at high curvatures is fundamental to its biological activity, yet this regime has been difficult to probe experimentally, and literature results have not been consistent. We created a ‘molecular vise’ in which base-pairing interactions generated a compressive force on sub-persistence length segments of dsDNA. Short dsDNA strands (<41 base pairs) resisted this force and remained straight; longer strands became bent, a phenomenon called ‘Euler buckling’. We monitored the buckling transition via Förster Resonance Energy Transfer (FRET) between appended fluorophores. For low-to-moderate concentrations of monovalent salt (up to ∼150 mM), our results are in quantitative agreement with the worm-like chain (WLC) model of DNA elasticity, without the need to invoke any ‘kinked’ states. Greater concentrations of monovalent salts or 1 mM Mg2+ induced an apparent softening of the dsDNA, which was best accounted for by a kink in the region of highest curvature. We tested the effects of all single-nucleotide mismatches on the DNA bending. Remarkably, the propensity to kink correlated with the thermodynamic destabilization of the mismatched DNA relative the perfectly complementary strand, suggesting that the kinked state is locally melted. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA.

DNA kinks and bubbles: Temperature dependence of the elastic energy of sharply bent 10-nm-size DNA molecules

A 10-nm-long DNA molecule can bend through large angles reversibly. Past the linear regime, its equilibrium nonlinear bending elasticity is governed by a critical bending torque τ(c)≈30pN×nm at which the molecule develops a kink. This nonlinearity has long been attributed to the nucleation of a bubble or melted region in the molecule. Here we measure the temperature dependence of the critical bending torque for nicked DNA, and determine that the entropy associated with the kink in the nonlinear regime is negligible. Thus in the case of nicked DNA the kink is not a bubble, but a compact region deformed beyond a yield strain. We further argue that, with our boundary conditions, the same is likely true for intact DNA. The present measurements confirm that the critical bending torque τ(c) is a materials parameter of DNA mechanics analogous to the bending modulus B≈200pN×nm.

Atomistic simulations of nucleosomes

AbstractNearly a dozen all‐atom molecular dynamics (MD) simulations of the nucleosome have been performed. Collectively, these simulations provide insights into the structure and dynamics of the biomolecular complex that serves as the fundamental folding unit of chromatin. Nucleosomes contain 146 base pairs of DNA wrapped in a left‐handed superhelix around a core of eight histones. This review provides a survey of what has been learned about DNA, histones, and solvent interactions based on all‐atom MD studies of the nucleosome. The longest simulations to date are on the order of 100 nanoseconds. On this time scale, nucleosomes are quite stable. DNA kinks, the histone tails, solvent, and ions are highly dynamic and can be readily investigated using equilibrium dynamics methods. Steered MD is required to observe large‐scale structural changes. The need for explicit solvent techniques is underscored by the inability of continuum solvent methods to properly describe the ion‐nucleosome radial distribution functions. The atomistic techniques reviewed here are deemed necessary for exploration of the near infinite variations in atomic composition that exists even in the canonical nucleosome octamer. Continued development of these nascent simulation efforts will enable experimentalists to utilize rational design strategies in their efforts to investigate nucleosomes and chromatin. © 2013 John Wiley &amp; Sons, Ltd.This article is categorized under: Molecular and Statistical Mechanics &gt; Molecular Dynamics and Monte-Carlo Methods

113 Conformational dynamics of human 8-oxoguanine-DNA glycosylase

DNA continuously undergoes oxidation damage from both exogenous and endogenous sources, including ionizing radiation, ultraviolet light, and products of metabolism. Replication of damaged DNA sometimes gives rise to mutations which can contribute to disease and aging. One of the most mutagenic lesions caused by DNA oxidation is 7,8-dihydro-8-oxoguanine (oxoG), which, if not repaired, results in G → T transversions. In human cells, oxoG is repaired through excision by 8-oxoguanine-DNA glycosylase hOGG1. In addition to its glycosylase activity, hOGG1 possesses an AP-lyase activity, which catalyzes the elimination of the 3’-phosphate (β-elimination) at the nascent, or preformed abasic (AP) site. The glycosidic bond breakage is initiated by a nucleophilic attack at C1’ by the Lys-249 residue resulting in a covalent enzyme–DNA-Schiff base intermediate, which then rearranges, and undergoes elimination. The 3-D structure of hOGG1shows that DNA binding is accompanied with drastic conformational changes, including DNA kinking, eversion of oxoGua from the double helix, and insertion of few amino acid residues into DNA. Previously (Kuznetsov et al., 2005, 2007), we have studied the stopped-flow kinetics of oxoG and AP site lesions processing by hOGG1. The character of tryptophan and 2-aminopurine fluorescence traces revealed that both the protein and the damaged DNA undergo extensive conformational changes in the course of DNA substrate binding- and -cleavage. To understand better, the mechanism by which hOGG1 recognizes DNA lesions, we have examined the influence of amino acid substitutions on conformational dynamics of hOGG1 and DNA during specific site recognition and conversion. Fluorescence kinetics of enzyme mutant forms F45 W, F319 W, Y203 W, Y203A, H270 W, K249Q demonstrated the multistep character of catalytic process and made clear the role of these amino acids for hOGG1 catalysis.

Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A.

High-mobility group B (HMGB) proteins bind duplex DNA without sequence specificity, facilitating the formation of compact nucleoprotein structures by increasing the apparent flexibility of DNA through the introduction of DNA kinks. It has remained unclear whether HMGB binding and DNA kinking are simultaneous and whether the induced kink is rigid (static) or flexible. The detailed molecular mechanism of HMGB-induced DNA ‘softening’ is explored here by single-molecule fluorescence resonance energy transfer studies of single yeast Nhp6A (yNhp6A) proteins binding to short DNA duplexes. We show that the local effect of yNhp6A protein binding to DNA is consistent with formation of a single static kink that is short lived (lifetimes of a few seconds) under physiological buffer conditions. Within the time resolution of our experiments, this static kink occurs at the instant the protein binds to the DNA, and the DNA straightens at the instant the protein dissociates from the DNA. Our observations support a model in which HMGB proteins soften DNA through random dynamic binding and dissociation, accompanied by DNA kinking and straightening, respectively.

Physical Organization of DNA by Multiple Non-Specific DNA-Binding Modes of Integration Host Factor (IHF)

The integration host factor (IHF) is an abundant nucleoid-associated protein and an essential co-factor for phage λ site-specific recombination and gene regulation in E. coli. Introduction of a sharp DNA kink at specific cognate sites is critical for these functions. Interestingly, the intracellular concentration of IHF is much higher than the concentration needed for site-specific interactions, suggesting that non-specific binding of IHF to DNA plays a role in the physical organization of bacterial chromatin. However, it is unclear how non-specific DNA association contributes to DNA organization. By using a combination of single DNA manipulation and atomic force microscopy imaging methods, we show here that distinct modes of non-specific DNA binding of IHF result in complex global DNA conformations. Changes in KCl and IHF concentrations, as well as tension applied to DNA, dramatically influence the degree of DNA-bending. In addition, IHF can crosslink DNA into a highly compact DNA meshwork that is observed in the presence of magnesium at low concentration of monovalent ions and high IHF-DNA stoichiometries. Our findings provide important insights into how IHF contributes to bacterial chromatin organization, gene regulation, and biofilm formation.

Conformational dynamics of the interaction of Escherichia coli endonuclease VIII with DNA substrates

Endonuclease VIII (Nei) from Escherichia coli is a DNA repair enzyme that removes a wide range of oxidized pyrimidine bases from DNA. As inferred from the crystal structures and biochemical studies, recognition of DNA lesions by Nei involves several conformational changes in both protein and DNA, such as DNA kinking, damaged base eversion into the enzyme's active site, and insertion of a loop of the enzyme into the void formed by the eversion. Excision of the damaged base by Nei also proceeds through several chemical steps: N-glycosidic bond breakage, β-elimination and δ-elimination of the phosphates flanking the lesion. We have used stopped-flow kinetics with fluorescence detection to follow conformational changes in the Nei molecule when the enzyme binds normal DNA, damaged but uncleavable DNA, or several cleavable damaged DNA substrates. Binding normal or damaged uncleavable DNA proceeded in two fluorescently discernible reversible stages, while processing of cleavable substrates involved three reversible stages followed by and irreversible stage and equilibrium with the reaction product. Individual rate constants were calculated for each reaction step. Based on the stopped-flow data, crystal structure, and a comparison with the stopped-flow kinetics of E. coli formamidopyrimidine-DNA glycosylase, a homolog of Nei, we propose the nature of some of the steps that may be involved into the recognition and excision of damaged bases by Nei.

Insights into the thermal stabilization and conformational transitions of DNA by hyperthermophile protein Sso7d: molecular dynamics simulations and MM-PBSA analysis

In the assembly of DNA-protein complex, the DNA kinking plays an important role in nucleoprotein structures and gene regulation. Molecular dynamics (MD) simulations were performed on specific protein-DNA complexes in this study to investigate the stability and structural transitions of DNA depending on temperature. Furthermore, we introduced the molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) approach to analyze the interactions between DNA and protein in hyperthermophile. Focused on two specific Sso7d-DNA complexes (PDB codes: 1BNZ and 1BF4), we performed MD simulations at four temperatures (300, 360, 420, and 480 K) and MM-PBSA at 300 and 360 K to illustrate detailed information on the changes of DNA. Our results show that Sso7d stabilizes DNA duplex over a certain temperature range and DNA molecules undergo B-like to A-like form transitions in the binary complex with the temperature increasing, which are consistent with the experimental data. Our work will contribute to a better understanding of protein-DNA interaction.

Динамические характеристики кинков и антикинков ДНК

Динамические характеристики кинков и антикинков ДНК

Mapping the Transition State for DNA Bending by IHF

How DNA-bending proteins recognize their specific sites on DNA remains elusive, particularly for proteins that use indirect readout, which relies on sequence-dependent variations in DNA flexibility/bendability. The question remains as to whether the protein bends the DNA (protein-induced bending) or, alternatively, “prebent” DNA conformations are thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates and, in particular, snapshots of the transition state along the recognition pathway. We present such a snapshot, from measurements of DNA bending dynamics in complex with Escherichia coli integration host factor (IHF), an architectural protein that bends specific sites on λ-DNA in a U-turn by creating two sharp kinks in DNA. Fluorescence resonance energy transfer measurements in response to laser temperature-jump perturbation monitor DNA bending. We find that nicks or mismatches that enhance DNA flexibility at the site of the kinks show 3- to 4-fold increase in DNA bending rates that reflect a 4- to 11-fold increase in binding affinities, while sequence modifications away from the kink sites, as well as mutations in IHF designed to destabilize the complex, have negligible effect on DNA bending rates despite >250-fold decrease in binding affinities. These results support the scenario that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional protein-induced bending occurring after the transition state.

A Protein Biosensor That Relies on Bending of Single DNA Molecules

A "bendy" protein sensor: A DNA-based sensor that uses folded DNA (through DNA kinks) and protein-induced bending to detect DNA-binding proteins is presented. Single-molecule sensing of a transcriptional activator (catabolite activator protein, CAP, which bends its DNA site by 80°) is demonstrated in solution and on surfaces, both in buffers and in cell lysates. The method should allow detection of a wide range of DNA-bending proteins.

Influence of periodic field with constant and slowly changing frequency on movement of DNA kink

The movement of the kink in homogeneous polynucleotide chains of DNA under the influence of three kinds of an external periodic field--with constant, growing and decreasing frequencies is investigated in the work. Dependences of speed and coordinate of the kink from time have been found with the help of McLaughlin and Scott energetic method. Phase trajectories of the kink have been constructed. It has been shown that in the case of external action with constant frequency, the kink aspires to a limiting cycle; in the case of an external field with increasing frequency it tends to a stationary state corresponding to a special point of stable focus type; in the case of an external field with decreasing frequency, the kink transfers from one stationary state (unstable focus) to another (stable focus). The obtained results show the possibility of governing the kink movement by slowly changing frequency.