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DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.

The Bloom’s helicase ortholog, Sgs1, orchestrates the formation and disengagement of DNA joint molecules to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that akin to the Mus81-Mms4, Yen1 and MutLγ-Exo1 nucleases, Sgs1 helicase function is under tight cell cycle control through the actions of CDK and Polo kinase Cdc5. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M-phase, they collaboratively delimit Sgs1 activation to S-phase. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a general mechanism that drives orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

Magnetic tweezers provide a versatile toolkit supporting the mechanistic investigation of helicases. In the present article, we show that custom magnetic tweezers setups are straightforward to construct and can easily be extended to provide adaptable platforms, capable of addressing a multitude of enquiries regarding the functions of these fascinating molecular machines. We first address the fundamental components of a basic magnetic tweezers scheme and review some previous results to demonstrate the versatility of this instrument. We then elaborate on several extensions to the basic magnetic tweezers scheme, and demonstrate their applications with data from ongoing research. As our methodological overview illustrates, magnetic tweezers are an extremely useful tool for the characterization of helicases and a custom built instrument can be specifically tailored to suit the experimenter’s needs.

Summary The Bloom’s helicase ortholog, Sgs1, orchestrates the formation and disengagement of recombination intermediates to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that, akin to the Mus81-Mms4, Yen1, and MutLγ-Exo1 nucleases, Sgs1 helicase function is under cell-cycle control through the actions of CDK and Cdc5 kinases. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M phase, they act in concert to stimulate Sgs1 activity during S phase/prophase I. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a concerted mechanism driving orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

Magnetic tweezers are a wide-spread tool used to study the mechanics and the function of a large variety of biomolecules and biomolecular machines. This tool uses a magnetic particle and a strong magnetic field gradient to apply defined forces to the molecule of interest. Forces are typically quantified by analyzing the lateral fluctuations of the biomolecule-tethered particle in the direction perpendicular to the applied force. Since the magnetic field pins the anisotropy axis of the particle, the lateral fluctuations follow the geometry of a pendulum with a short pendulum length along and a long pendulum length perpendicular to the field lines. Typically, the short pendulum geometry is used for force calibration by power-spectral-density (PSD) analysis, because the movement of the bead in this direction can be approximated by a simple translational motion. Here, we provide a detailed analysis of the fluctuations according to the long pendulum geometry and show that for this direction, both the translational and the rotational motions of the particle have to be considered. We provide analytical formulas for the PSD of this coupled system that agree well with PSDs obtained in experiments and simulations and that finally allow a faithful quantification of the magnetic force for the long pendulum geometry. We furthermore demonstrate that this methodology allows the calibration of much larger forces than the short pendulum geometry in a tether-length-dependent manner. In addition, the accuracy of determination of the absolute force is improved. Our force calibration based on the long pendulum geometry will facilitate high-resolution magnetic-tweezers experiments that rely on short molecules and large forces, as well as highly parallelized measurements that use low frame rates.

Shadoo (Sho) is a neuronally expressed glycoprotein of unknown function. Although there is no overall sequence homology to the cellular prion protein (PrP(C)), both proteins contain a highly conserved internal hydrophobic domain (HD) and are tethered to the outer leaflet of the plasma membrane via a C-terminal glycosylphosphatidylinositol anchor. A previous study revealed that Sho can reduce toxicity of a PrP mutant devoid of the HD (PrPΔHD). We have now studied the stress-protective activity of Sho in detail and identified domains involved in this activity. Like PrP(C), Sho protects cells against physiological stressors such as the excitotoxin glutamate. Moreover, both PrP(C) and Sho required the N-terminal domain for this activity; the stress-protective capacity of PrPΔN as well as ShoΔN was significantly impaired. In both proteins, the HD promoted homodimer formation; however, deletion of the HD had different effects. Although ShoΔHD lost its stress-protective activity, PrPΔHD acquired a neurotoxic potential. Finally, we could show that the N-terminal domain of PrP(C) could be functionally replaced by that of Sho, suggesting a similar function of the N termini of Sho and PrP(C). Our study reveals a conserved physiological activity between PrP(C) and Sho to protect cells from stress-induced toxicity and suggests that Sho and PrP(C) might act on similar signaling pathways.

Twisting a DNA molecule held under constant tension is accompanied by a transition from a linear to a plectonemic DNA configuration, in which part of the applied twist is absorbed in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. To elucidate its origin we study this abrupt DNA shortening using magnetic tweezers. We find that it strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. By quantitative data analysis, relevant plectoneme energies and other parameters were extracted, providing good agreement with a simple theory. As a direct confirmation of the initial-loop model, we find that for a kinked DNA molecule the abrupt extension change occurs at significantly lower twist than the subsequent superhelix formation. This should allow pinning of the plectoneme position within supercoiled DNA if a kinked substrate is used, and enable the detection of enzymes and proteins which, themselves, bend or kink DNA.

The type I restriction–modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrP(Palm)) via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells. These latter results enable experiments on the cellular level to elucidate the mechanism and site of PrP-PrP(Sc) conversion.

CRISPR-Cas systems provide adaptive immunity against invading genetic elements such as phages and plasmids by degrading the invader DNA. In type I CRISPR-Cas systems the intrinsic RNA component of the Cascade effector complex recognizes a complementary DNA target strand (protospacer) through base pairing while displacing the non-target strand of the duplex. The resulting RNA-DNA hybrid is called R-loop. R-loop establishment recruits the helicase-nuclease Cas3, which subsequently degrades the target DNA. Here we use single-molecule DNA supercoiling experiments to investigate the recognition and verification of the target sequence by Cascade. To this end we carefully explore the effect of mutations across the target. We observe the occurrence of intermediate R-loops which lengths correspond exactly to the distance of the mutation from the start of the target sequence. These intermediate R-loops are unstable and their stability directly correlates with their length. When point mutations are eventually overcome, a full R-loop forms and becomes “locked” through conformational changes of the Cascade complex. These observations provide direct evidence for a directional R-loop formation and suggest a dynamic target scanning by Cascade, mediated through R-loop intermediates. Early encountered mutations challenge R-loop propagation and cause its collapse while distal mutations enable more stable and longer lived R-loops to overcome them. The target validation takes place exclusively at the end of the sequence when full R-loop formation induces conformational changes that initiate DNA degradation. Such a dynamic and directional R-loop propagation scheme offers several advantages for suitable homologous target recognition: (i) the complex spends little time on wrong targets that from the beginning on carry mismatches; (ii) however it supports that point mutations can be tolerated thus precluding invading DNA to escape degradation through protospacer mutations.

ClpB belongs to the Hsp100 family and assists de-aggregation of protein aggregates by DnaK chaperone systems. It contains two Walker consensus sequences (or P-Loops) that indicate potential nucleotide binding domains (NBD). Both domains appear to be essential for chaperoning function, since mutation of the conserved lysine residue of the GX4GKT consensus sequences to glutamine (K204Q and K601Q) abolishes its properties to accelerate renaturation of aggregated firefly luciferase. The underlying biochemical reason for this malfunction appears not to be a dramatically reduced ATPase activity of either P-loop per se but rather changed properties of co-operativity of ATPase activity connected to oligomerization properties to form productive oligomers. This view is corroborated by data that show that structural stability (as judged by CD spectroscopy) or ATPase activity at single turnover conditions (at low ATP concentrations) are not significantly affected by these mutations. In addition nucleotide binding properties of wild-type protein and mutants (as judged by binding studies with fluorescent nucleotide analogues and competitive displacement titrations) do not differ dramatically. However, the general pattern of formation of stable, defined oligomers formed as a function of salt concentration and nucleotides and more importantly, cooperativity of ATPase activity at high ATP concentrations is dramatically changed with the two P-loop mutants described.

The Escherichia coli DnaK (DnaKEco) chaperone cycle is tightly regulated by the cochaperones DnaJ, which stimulates ATP hydrolysis, and GrpE, which acts as a nucleotide exchange factor. The Thermus thermophilus DnaK (DnaKTth) system additionally comprises the DnaK-DnaJ assembly factor (DafATth) that is mediating formation of a 300 kDa DnaKTth. DnaJTth.DafATth complex.A model peptide derived from the tumor suppressor protein p53 was used to dissect the regulation of the individual kinetic key steps of the DnaKTth nucleotide/chaperone cycle. As with DnaKEco the DnaKTth.ATP complex binds substrates with reduced affinity and large exchange rates compared to the DnaKTth.ADP.Pi state. In contrast to DnaKEco, ADP-Pi release is slow compared to the rate of hydrolysis, reversing the balance of the two functional nucleotide states. Whereas GrpETth stimulates nucleotide release from DnaKTth, DnaJTth does not accelerate ATP hydrolysis under various experimental conditions. However, it exerts influence on the interaction of DnaKTth with substrates: in the presence of DafATth, DnaJTth inhibits substrate binding, and substrate already bound to DnaKTth is displaced by DnaJTth and DafATth, indicating competitive binding of DnaJTth/DafATth and substrate. It thus appears that the DnaKTth. DnaJTth.DafATth complex as isolated from T. thermophilus does not represent the active species in the DnaKTth chaperone cycle. Isothermal titration calorimetry showed that the ternary complex of DnaKTth, DnaJTth and DafATth is assembling with high affinity, whereas binary complexes of DnaKTth and DnaJTth or DafATth were not detectable, indicating highly synergistic formation of the 300 kDa DnaKTth. DnaJTth.DafATth complex. Based on these results, a model describing the DnaKTth chaperone cycle and its regulation by cochaperones is proposed where DnaKTth. DnaJTth.DafATth constitutes the resting state, and a DnaKTth. substrate.DnaJTth complex is the active chaperone species. The novel factor DafATth that mediates interaction of DnaKTth with DnaJTth would thus serve as a "template" to stabilise the ternary DnaKTth.DafATth.DnaJTth complex until it is replaced by substrate proteins under heat shock conditions.

During the last decade, DNA origami has become a powerful tool in research at the nanoscale. The relative ease of constructing functionalized DNA origami structures of a defined shape allows for their applications in membrane biophysics. Recently, we have constructed stiff rod-like DNA origami structures consisting of six DNA helixes, which were functionalized with hydrophobic membrane-binding anchors and fluorescently labeled at defined positions [1]. Selective fluorescent labeling allowed us to determine the translational and rotational diffusion coefficients of the DNA origami rods on lipid membranes by fluorescence correlation spectroscopy, which were found to be in a good agreement with the hydrodynamics-based theory of membrane diffusion. Further, we studied the effect of the surface density of membrane-bound origami structures on their Brownian motion and found a strong decrease of the translational and rotational diffusion coefficients of membrane-bound nanorods with an increase in their surface density. We compare our experimental findings with results of Monte Carlo simulations of Brownian hard needles in 2D.[1] A. Czogalla, E. P. Petrov, D. J. Kauert, V. Uzunova, Y. Zhang, R. Seidel, and P. Schwille, Faraday Discuss.161 (2013) 31.

Restriction enzymes (REs) are the central part of the defence system of bacteria against invading viruses. The protein complexes recognize viral DNA by the methylation state of their target sequence and destroy it by cleaving it into pieces. For this, the majority of REs need to interact with two distant target sites. This long-range inter-site communication can be accomplished either by passive 3D diffusive looping or by 1D motion along the DNA contour. Among the different classes of REs, Type I and Type III REs play a special role due to their helicase domains, which are key to the inter-site communication.For Type I REs it is well established that the helicase domain acts as a dsDNA translocating motor. Cleavage is triggered after a pure 1D communication process, when two translocating motors from distant target sites collide.In comparison, the communication mechanism for Type III REs has not been accurately defined and conflicting models including 3D diffusion and 1D translocation have been proposed. Using single-molecule DNA stretching based on magnetic tweezers, we provide evidence for a pure 1D communication mechanism in the absence of any 3D diffusive looping. Furthermore, we exclude translocation for inter-site communication due to the low ATPase rates and the observation that the enzymes move bidirectionally along DNA. From this we conclude that Type III REs use 1D diffusion to communicate between their distant target sites.In order to test the diffusion hypothesis we have started to track the movement of Type I and III REs along DNA using a setup combining magnetic tweezers with single-molecule fluorescence (total-internal reflection fluorescence microscopy).

Friction within biomolecules has recently gained increasing interest. Here we present a method that allows to study the friction that occurs during fast, few nm-sized refolding processes of nucleic acids. Branch migration of a homologous Holliday junction serves as an ideal system where such friction can be investigated. In this four-arm DNA junction the opposing arms possess identical sequences with respect to the junction center. In the absence of external constraints the junction is mobile such that one pair of homologous arms can expand at the expense of the other in single base pair diffusive steps. We measure the dynamics of the branch migration process by stretching a torsionally constrained Holliday junction using magnetic tweezers and measuring the length fluctuations of the arms with high-speed videomicroscopy at ∼3 kHz. Since DNA has a helical structure, branch migration causes twisting of the arms with one turn per helical pitch moved. This constrains the movement of the junction within the tweezers to ∼10 bp. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Thus they cannot be directly resolved. However, power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Theoretical modeling considering the elastic coupling of DNA bending fluctuations and the junction movement allows to quantitatively determine the stepping rate and thus the friction of the branch migration process. We expect that our method is widely applicable to study local-scale molecular friction in biological systems.

The recent development of the origami technique has revolutionized DNA-based assembly by allowing the synthesis of arbitrarily shaped and sub-micrometer sized two-dimensional (2D) and three-dimensional (3D) architectures with atomic precision. Such structures offer great potential for the development of nanomechanical elements, mediators and sensors, which however requires detailed understanding of their complex mechanical properties. Using magnetic tweezers, we here present direct mechanical measurements on single DNA origami structures and characterize the bending and torsional rigidity of four- and six-helix bundles assembled by this technique. Compared to duplex DNA, we find the bending rigidities to be greatly increased while the torsional rigidities are only moderately augmented. We present a mechanical model explicitly including the crossovers between the individual helices in the origami structure that can describe the experimentally observed behavior. Our results provide an important basis for future applications of 3D DNA origami structures as rigid scaffolds and force transducers as well as noise suppressors in single-molecule mechanical measurements. Beyond that we show how origami structures can be defined and rigidly interfaced to surfaces, which is an important prerequisite to develop structured and three-dimensional surface modifications with the help of DNA templates.

The worm-like-chain (WLC) model is widely used to describe the energetics of DNA bending. However, for length scales much shorter than the persistence length the validity of a purely harmonic bending potential has been questioned by recent experiments. So-called sub-elastic chain (SEC) models were proposed that predict a lower elastic energy of highly bent DNA conformations. Until now, no unambiguous verification of these models has been obtained since probing the elasticity of DNA on short length scales remains challenging. Here we address this issue by modeling single molecule supercoiling experiments of DNA under tension using coarse-grained Monte Carlo simulations. Twisting of stretched DNA is accompanied by an abrupt decrease of the DNA extension at a critical supercoil density due to buckling of the molecule. This transition is caused by an energetic offset due to the strongly bent end-loop of the forming superhelical structure. While simulations based on WLC bending energetics could quantitatively reproduce the buckling measured in magnetic tweezers experiments, the buckling almost disappears for the tested linear SEC model. Thus, our data support the validity of a harmonic bending potential even for strongly bent DNA down to bending radii of 3.5 nm.

Total internal reflection fluorescence (TIRF) microscopy is an elegant optical technique that limits the dimension of the volume, in which fluorophores are excited, along the z-direction to the hundred nanometer-scale. The method makes use of the evanescent field, which arises when light is totally internally reflected at the boundary to a medium of lower refractive index. Often the penetration depth of this exponentially decaying field is left undetermined, which sets limits to the reproducibility in different experiments. Here, we present a novel method to measure this quantity: We use a quantum dot as a point-like light source and a Holliday junction as a drive to move the fluorescent probe with nanometer precision perpendicular to the substrate surface. The junction serves as a worm drive, which couples rotation into translational movement, while the DNA pitch serves as a precise intrinsic ruler. The Holliday junction is forced to migrate by adding negative turns to the DNA stretched perpendicular to the surface using magnetic tweezers. This causes the quantum dot, which is attached upstream of the junction, to decrease its height above the surface by 3.4 nm per turn. Thus it can be moved continuously through the excitation field while monitoring its height-dependent fluorescent signal. Since the quantum dot is a point-like light source, the intensity decay of the evanescent field can be obtained by dividing the signal recorded in TIRF illumination by the one recorded in conventional epi-illumination without further corrections. Our assay should allow for probing the axial intensity profile of any electromagnetic field distribution with a point-like light source, without the necessity of attaching it to an interfering surface.

Type III restriction enzymes sense the relative orientation of their distant target sites and cleave DNA only if at least two of them are situated in an inverted repeat. The communication process is strictly dependent on ATP hydrolysis catalyzed by their superfamily 2 helicase domains. Given the similarity to Type I restriction enzymes, which couple ATP hydrolysis to directed motion on DNA, unidirectional loop translocation that may partially be accompanied by 3D diffusive looping has been the suggested communication mechanism for Type III enzymes.Based on magnetic tweezers single-molecule cleavage experiments and ATPase measurements we suggest an alternative inter-site communication mechanism using 1D diffusion along the DNA contour (1). In order to verify this hypothesis we directly visualize the motion of quantum-dot labeled Type III restriction enzymes along DNA. For this we use a setup that combines magnetic tweezers with total internal reflection fluorescence microscopy. The enzymes undergo a fast diffusive motion along DNA capable of scanning kbp distances per second. We also find that the affinity of the enzymes to non-specific and specific DNA is regulated by the presence of ATP suggesting that ATP hydrolysis acts as a trigger for diffusion.Thus Type III restriction enzymes are the first DNA-modifying enzymes which communicate target site orientations over long distances via 1D diffusion.(1) van Aelst K, Toth J, Ramanathan SP, Schwarz FW, Seidel R, Szczelkun MD. Proc Natl Acad Sci U S A. 2010 May 18;107(20):9123-8.