DNA Contour Research Articles

Human DNA mismatch repair: coupling of mismatch recognition to strand-specific excision.

Eukaryotic mismatch-repair (MMR) proteins MutSα and MutLα couple recognition of base mismatches to strand-specific excision, initiated in vivo at growing 3′ ends and 5′ Okazaki-fragment ends or, in human nuclear extracts, at nicks in exogenous circular substrates. We addressed five biochemical questions relevant to coupling models. Excision remained fully efficient at DNA:MutSα ratios of nearly 1 to 1 at various mismatch-nick distances, suggesting a requirement for only one MutSα molecule per substrate. As the mismatch-nick DNA contour distance D in exogenous substrates increased from 0.26 to 0.98 kbp, initiation of excision in extracts decreased as D−0.43 rather than the D−1 to D−2 predicted by some translocation or diffusion models. Virtually all excision was along the shorter (3′–5′) nick-mismatch, even when the other (5′–3′) path was less than twice as long. These observations argue against stochastically directed translocating/diffusing recognition complexes. The failure of mismatched DNA in trans to provoke excision of separate nicked homoduplexes argues against one-stage (concerted) triggering of excision initiation by recognition complexes acting through space. However, proteins associated with gapped DNA did appear to compete in trans with those in cis to mismatch-associated proteins. Thus, as in Escherichia coli, eukaryotic MMR may involve distinct initial-activation and excision-path-commitment stages.

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1-3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 +/- 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.

DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.

Structural analysis of hyperperiodic DNA from Caenorhabditis elegans

Several bioinformatics studies have identified an unexpected but remarkably prevalent ∼10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.

Ss-LrpB from Sulfolobus solfataricus Condenses about 100 Base Pairs of Its Own Operator DNA into Globular Nucleoprotein Complexes

Ss-LrpB from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2 is a member of the Lrp-like family of Bacterial/Archaeal transcription regulators that binds its own control region at three regularly spaced and partially conserved 15-bp-long imperfect palindromes. We have used atomic force microscopy to analyze the architecture of Ss-LrpB.DNA complexes with a different stoichiometry formed with the wild type operator and with an operator mutant. Binding of dimeric Ss-LrpB to all three target sites is accompanied by the formation of globular complexes, in which the protein induces strong DNA deformations. Furthermore, DNA contour length foreshortening of these complexes indicates DNA wrapping, with about 100 bp being condensed. The average bending angle is 260 degrees . The establishment of protein-protein contacts between Ss-LrpB dimers in these globular complexes will contribute to the cooperativity of the binding. The profound remodeling of the control region is expected to have a strong impact on gene expression and might constitute the key element in the autoregulatory process.

Amine terminated G-6 PAMAM dendrimer and its interaction with DNA probed by Hoechst 33258

Fluorescence characteristics of Hoechst 33258 bound to G-6 dendrimer, to the DNA–G-6 dendrimer complex, and to DNA were compared with that in an aqueous solution. The spectral properties including fluorescence emission spectrum, accessibility of anionic quencher, as well as the fluorescence decay time of the Hoechst 33258 are different for all three conditions, indicating that the environments in these conditions are different. Close analysis of the fluorescence properties led us to suggest that Hoechst 33258 located at or near the contact area of the dendrimer and DNA in the DNA–G-6 complex. In the complex, in the absence of Hoechst 33258, the shape of the circular dichroism in the DNA absorption region remained, indicating that DNA is in B form in the complex. On the other hand, the magnitude of linear dichroism (LD) decreased upon DNA–G-6 dendrimer complex formation. The decrease in LD magnitude reflects the shortening of the DNA contour length, which is expected from the fact that a large part of linear DNA is required to wrap the surface of G-6 dendrimer.

Mapping the Phase Diagram of Single DNA Molecule Force-Induced Melting in the Presence of Ethidium

When a single DNA molecule is stretched beyond its normal contour length, a force-induced melting transition is observed. Ethidium binding increases the DNA contour length, decreases the elongation upon melting, and increases the DNA melting force in a manner that is consistent with the ethidium-induced changes in duplex DNA stability known from thermal melting studies. The DNA stretching curves map out a phase diagram and critical point in the force-extension-ethidium concentration space. Intercalation occurs between alternate base pairs at low forces and between every base pair at high forces.

Dual Binding Modes for an HMG Domain from Human HMGB2 on DNA

High mobility group B (HMGB) proteins contain two HMG box domains known to bind without sequence specificity into the DNA minor groove, slightly intercalating between basepairs and producing a strong bend in the DNA backbone. We use optical tweezers to measure the forces required to stretch single DNA molecules. Parameters describing DNA flexibility, including contour length and persistence length, are revealed. In the presence of nanomolar concentrations of isolated HMG box A from HMGB2, DNA shows a decrease in its persistence length, where the protein induces an average DNA bend angle of 114 ± 21° for 50 mM Na +, and 87 ± 9° for 100 mM Na +. The DNA contour length increases from 0.341 ± 0.003 to 0.397 ± 0.012 nm per basepair, independent of salt concentration. In 50 mM Na +, the protein does not unbind even at high DNA extension, whereas in 100 mM Na +, the protein appears to unbind only below concentrations of 2 nM. These observations support a flexible hinge model for noncooperative HMG binding at low protein concentrations. However, at higher protein concentrations, a cooperative filament mode is observed instead of the hinge binding. This mode may be uniquely characterized by this high-force optical tweezers experiment.

The CSB Protein Actively Wraps DNA

The CSB protein is a member of the SWI2/SNF2 family of ATP-dependent chromatin remodeling factors and is essential for transcription-coupled DNA repair. The role of CSB in this DNA repair process is unclear, but the protein was found to remodel nucleosomes and alter DNA double helix conformation upon binding. Elucidating the nature of the change in DNA structure induced by CSB is of great interest for understanding the CSB mechanism of action. We analyzed the CSB.DNA complex by scanning force microscopy and measured a shortening of DNA contour length upon CSB binding in the presence of ATP. This DNA length reduction most likely results from DNA wrapping around the protein. Shorter DNA molecules were observed more frequently in the presence of non-hydrolyzable ATP analogues. These results suggest that DNA wrapping depends on ATP binding, whereas ATP hydrolysis results in unwrapping. We also provide evidence suggesting that CSB binds DNA as a dimer. DNA wrapping and unwrapping allows CSB to actively alter the DNA double helix conformation, which could influence nucleosomes and other protein-DNA interactions.

Interactive measurement and characterization of DNA molecules by analysis of AFM images

In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 +/- 5 nm and 910 +/- 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of lambda-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 +/- 5 nm) is consistent with values measured by other imaging techniques. An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible.

Initiation of translocation by Type I restriction-modification enzymes is associated with a short DNA extrusion

Recognition of 'foreign' DNA by Type I restriction-modification (R-M) enzymes elicits an ATP-dependent switch from methylase to endonuclease activity, which involves DNA translocation by the restriction subunit HsdR. Type I R-M enzymes are composed of three (Hsd) subunits with a stoichiometry of HsdR2:HsdM2:HsdS1 (R2-complex). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR1:HsdM2:HsdS1 (R1-complex). ATPS was used to trap initial translocation complexes, which were visualized by Atomic Force Microscopy (AFM). In the R1-complex, a small bulge, associated with a shortening in the contour-length of the DNA of 8 nm, was observed. This bulge was found to be sensitive to single-strand DNA nucleases, indicative of non-duplexed DNA. R2-complexes appeared larger in the AFM images and the DNA contour length showed a shortening of approximately 11 nm, suggesting that two bulges were formed. Disclosure of the structure of the first stage after the recognition-translocation switch of Type I restriction enzymes forms an important first step in resolving a detailed mechanistic picture of DNA translocation by SF-II DNA translocation motors.

Obstacle Bypass in Protein Motion along DNA by Two-dimensional Rather than One-dimensional Sliding

Site-specific DNA-binding proteins locate their target sites by facilitated diffusion. Several proteins have been shown to slide along DNA in vitro. However, whereas sliding is often envisaged as one-dimensional tracking of the DNA major groove, such a mechanism would not allow linear diffusion over long distances in vivo, where short stretches of free DNA are delimited by bound proteins. I propose a two-dimensional sliding mechanism, in which the protein diffuses freely on the cylindrical DNA surface, and I present experiments that can distinguish between one- and higher-dimensional diffusion along the DNA contour length. At 100 mm NaCl, translocation of EcoRI restriction endonuclease between sites on two DNA helices connected by a Holliday junction is as efficient as between sites on the same helix, indicating a three-dimensional mechanism. At 25 mm NaCl, translocation between sites on the same DNA helix is more efficient, indicating a role for sliding at low ionic strength. Obstacles attached to the major groove of one face of the DNA helix did not interfere with sliding, regardless of their orientation relative to the cleavage sites. This result is compatible with two-dimensional but not one-dimensional sliding. As illustrated by Monte-Carlo simulation, two-dimensional sliding may not only allow proteins to move around nucleosomes in vivo but also reduce the redundancy of their search for the target site.

Mode of drug binding to DNA determined by optical tweezers force spectroscopy

Optical tweezers were employed to investigate the effects of small DNA-binding molecules on the low-force (≤ 15 pN) stretching behaviour of single DNA molecules. As the canonical B-DNA helix is not perturbed in this force regime, the effects on DNA elasticity observed upon drug binding provide useful insight into how DNA-binding drugs may alter in vivo processes. In this study, the effects of agents with different DNA binding modes were analysed. DNA force—extension curves were recorded in the presence of netropsin, a purely minor groove-binding antibiotic drug, ethidium bromide, an intercalating fluorescent dye, and berenil, an antiprotozoal drug proposed to exhibit both intercalative and minor groove-binding modes. Applying an approximation of the worm-like chain model, which describes the low-force stretching behaviour, the results were analysed in terms of the DNA contour length and persistence length. From these single molecule studies it was observed that minor groove-binding and intercalating modes of DNA-binding could be distinguished based on changes to DNA elasticity.

HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study.

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.

Single DNA Molecule Analysis of Transcription Complexes

This chapter describes procedures developed to assemble, image, and analyze initiation and elongation transcription complexes of both bacterial and eukaryotic RNA polymerases (RNAP). The chapter describes basic methods for the conformational analysis of transcription complexes by means of atomic force microscopy. Buffer conditions for the equilibration of DNA and protein-DNA complexes on mica surface are first presented. Equilibration, as opposed to trapping onto the surface, is an essential prerequisite for a meaningful analysis, as only the structure of equilibrated molecules can be reliably related to their three-dimensional solution structure. Next methods to analyze the contour length of DNA molecules are presented. These are non-trivial image processing procedures that can significantly affect AFM measurements. As an example, it has been shown how a careful analysis of the DNA contour length of transcription complexes can give information on the extent of RNAP-DNA interaction. The determination of protein-induced DNA bend angles, using either tangents or the mean-square end-to-end distance, is then described. Finally, a novel DNA-Streptavidin tag method for the labeling of individual RNAP subunits is presented. The focus is on transcription, the methodology described here is of general practical use for imaging protein-DNA complexes by high-resolution microscopy.

Mode of drug binding to DNA determined by optical tweezers force spectroscopy

Abstract Optical tweezers were employed to investigate the effects of small DNA-binding molecules on the low-force (≤ 15 pN) stretching behaviour of single DNA molecules. As the canonical B-DNA helix is not perturbed in this force regime, the effects on DNA elasticity observed upon drug binding provide useful insight into how DNA-binding drugs may alter in vivo processes. In this study, the effects of agents with different DNA binding modes were analysed. DNA force—extension curves were recorded in the presence of netropsin, a purely minor groove-binding antibiotic drug, ethidium bromide, an intercalating fluorescent dye, and berenil, an antiprotozoal drug proposed to exhibit both intercalative and minor groove-binding modes. Applying an approximation of the worm-like chain model, which describes the low-force stretching behaviour, the results were analysed in terms of the DNA contour length and persistence length. From these single molecule studies it was observed that minor groove-binding and intercalating modes of DNA-binding could be distinguished based on changes to DNA elasticity.

Effect of glycerol-induced DNA conformational change on the separation of DNA fragments by capillary electrophoresis

The effect on DNA separation of adding glycerol to the running buffer was studied using linear polyacrylamide (LPA) or poly(ethylene oxide) (PEO) as separation medium. For both LPA and PEO, it was found that the addition of 25% (v/v) glycerol to the running buffer enhanced the separation of large double-stranded DNA fragments and increased the migration time. The two buffers used, 1×TBE (Tris–boric acid–EDTA) and 1×TTE (Tris– N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)–EDTA), showed similar improvement, but the effect on the 1×TBE buffer was more amplified. The difference in buffer properties, such as viscosity, conductance, and pH, had little effect on the separation. We attribute the improvements made in the separation to the ability of glycerol to induce a conformational change in DNA as demonstrated by dynamic light scattering results. The presence of glycerol can increase the electrostatic interactions between the phosphate groups, decrease the hydration sphere of the polynucleotides, and compete with water to form hydrogen bonds with the side group of bases. These interactions increase the DNA contour length and reduce the effective charge over weight ratio, which can explain the experimental data. The complex formed by boric acid and glycerol had a stronger effect on the DNA conformation change than glycerol itself. This enhancement was also observed in DNA sequencing analysis.

Diffusion-Controlled Intrachain Reactions of Supercoiled DNA: Brownian Dynamics Simulations

The Brownian Dynamics technique was used to model a diffusion-controlled intramolecular reaction of supercoiled DNA (2500 basepairs) in 0.1 M sodium chloride solution. The distance between the reactive groups along the DNA contour was 470 basepairs. The reaction radius was varied from 6 to 20 nm. The results are presented in terms of the probability distribution P F( t) of the first collision time. The general form of the function P F( t) could be correctly predicted by a simple analytical model of one-dimensional diffusion of the superhelix ends along the DNA contour. The distribution P F( t) is essentially non-exponential: within a large initial time interval, it scales as P F( t) ∼ t −1/2, which is typical for one-dimensional diffusion. However, the mean time of the first collision is inversely proportional to the reaction radius, as in three dimensions. A visual inspection of the simulated conformations showed that a considerable part of the collisions is caused by the bending of the superhelix axis in the regions of the end loops, where the axis is most flexible. This fact explains why the distribution P F( t) combines the features of one- and three-dimensional diffusion. The simulations were repeated for a DNA chain with a permanent bend of 100° in the middle position between the reactive groups along the DNA contour. The permanent bend changes dramatically the form of the distribution P F( t) and reduces the mean time of the first collision by approximately one order of magnitude.

Mutation in the Magnesium Binding Site of hMSH6 Disables the hMutSα Sliding Clamp from Translocating along DNA

In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutSalpha, a heterodimer of hMSH2 and hMSH6. In the presence of ATP and magnesium, hMutSalpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutSalpha variant, hMutSalpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with translocation. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The "translocation-prone" conformation of wild type hMutSalpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gammaS or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutSalpha in the presence and in the absence of magnesium helps explain the functional differences between hMutSalpha variants incapable of binding ATP as compared with those unable to bind the metal ion.

DNA bending due to specific p53 and p53 core domain-DNA interactions visualized by electron microscopy

We have used transmission electron microscopy to analyze the specificity and the extent of DNA bending upon binding of full-length wild-type human tumor suppressor protein p53 (p53) and the p53 core domain (p53CD) encoding amino acid residues 94–312, to linear double-stranded DNA bearing the consensus sequence 5′-AGACATGCCTAGACATGCCT-3′ (p53CON). Both proteins interacted with high specificity and efficiency with the recognition sequence in the presence of 50 mM KCl at low temperature (∼4 °C) while the p53CD also exhibits a strong and specific interaction at physiological temperature. Specific complex formation did not result in an apparent reduction of the DNA contour length. The interaction of p53 and the p53CD with p53CON induced a noticeable salt-dependent bending of the DNA axis. According to quantitative analysis with folded Gaussian distributions, the bending induced by p53 varied from ∼40 ° to 48 ° upon decreasing of the KCl concentration from 50 mM to ∼1 mM in the mounting buffer used for adsorption of the complexes to the carbon film surface. The p53CD bent DNA by 35–37 ° for all salt concentrations used in the mounting buffer. The bending angle of the p53/DNA complex under low salt conditions showed a somewhat broader distribution (σ≈39 °) than at high salt concentration (σ≈31 °) or for p53CD (σ≈24–27 °). Together, these results demonstrate that the p53CD has a dominant role in complex formation and that the complexes formed both by p53 and p53CD under moderate salt conditions are similar. However, the dependence of the bending parameters on ambient conditions suggest that the segments flanking the p53CD contribute to complex formation as well. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed.