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Sliding Restriction Helicase enzymes access the genetic information stored in double-helical DNA and RNA by opening the individual strands. Pseudo-helicases, including bacterial Type III restriction enzymes, use adenosine triphosphate (ATP) hydrolysis to communicate between two distant restriction sites on the same DNA and excise it only if the DNA is sensed as “foreign.” Schwarz et al. (p. 353 ) show that the bacterial Type III restriction enzyme, EcoP15I, undergoes an ATP-dependent conformational switch that promotes sliding along the DNA to allow the enzyme to localize to its target.

Recently, DNA origami became a powerful tool for custom-shaped functional biomolecules. In this paper, we present the first approach towards assembling amphipathic three-dimensional DNA origami nanostructures and assessing their dynamics on the surface of freestanding phospholipid membranes. Our nanostructures were stiff DNA origami rods comprising six DNA helices. They were functionalized with hydrophobic cholesteryl-ethylene glycol anchors and fluorescently labeled at defined positions. Having these tools in hand, we could demonstrate not only the capability of the amphipathic nanorods to coat membranes of various phospholipid compositions, but also their switchable liquid-ordered/liquid-disordered partitioning on phase separated membranes. The observed translocation of our nanostructures between different domains was controlled by divalent ions. Moreover, selective fluorescent labeling enabled us to distinguish between the translational and rotational diffusion of our six helix bundles on the membranes by fluorescence correlation spectroscopy. The obtained data reveal how DNA origami can be employed as a valuable tool in membrane biophysics.

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA. DOI: http://dx.doi.org/10.7554/eLife.18574.001

Native chemical ligation of unprotected peptides in organic solvents has been previously reported as a fast, efficient, and suitable method for coupling of hydrophobic peptides. However, it has not been determined whether the reaction can be carried out without possible side reactions or racemization. Here, we present a study on the chemoselectivity of this method by model reactions designed to test the reactivity of Arg and Lys side chains as well as that of α-amino groups. A possible racemization of the C-terminal amino acid of the N-terminal peptide was also investigated. The results show that ligation in organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups. Furthermore, no racemization of the C-terminal amino acid was observed if both educts were added simultaneously. Thus, native chemical ligation can be performed either in aqueous buffer systems or in organic solvents paving the way for the synthesis of larger hydrophobic peptides and/or membrane proteins. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

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.

Eukaryotic cell surface proteins are often modified by a glycosylphosphatidylinositol (GPI) anchor. More than 200 of these post-translationally altered proteins are presently known, a prominent example being the prion protein (PrP). Although the significance of the GPI anchor is well recognized, efforts to study its function are hampered due to its complex chemical nature, which combines hydrophilic glycosyl chains with hydrophobic lipid moieties. Here we describe a general method for the synthesis of a GPI-anchored peptide containing an N-terminal Cys. This module can be employed for the production of proteins containing a natural GPI anchor using expressed protein ligation.

Protein semisynthesis based on native chemical ligation has become a major protein engineering tool that allows manipulation of domains of proteins of all sizes. It helps to overcome limitations in chemical protein synthesis set by the inherent size limits of solid phase peptide synthesis. Here we present a semisynthesis approach that provides access to N-terminally-modified variants of human thymidine monophosphate kinase (TMPK). This enzyme is intimately involved in activating nucleoside-based drugs directed against viral infections such as HIV and against certain types of cancers. The option to chemically synthesize and manipulate the first 30 amino acids of this enzyme via protein semisynthesis allows direct substitution of vital amino acids in the P-loop of this enzyme for probing the mechanism of phosphate transfer and direct observation of substrate or inhibitor binding. Efficient native chemical ligation of two N-terminal segments, one comprising the wild type sequence and one containing a small fluorescent probe, provides milligram amounts of two semisynthetic TMPK variants. An efficient folding procedure in the presence of substrate nucleotides provides access to active semisynthetic TMPK variants.

Site-specific deposition of nanoparticles and DNA onto micro-patterned aminoterpolymer films is reported. The chemical patterning of the film surface is accomplished by an easy-to-handle, one-step procedure. Photolabile protection groups are locally removed by applying UV light through an optical mask. This causes exposure of amino groups to the surface to which charged nanoparticles can then associate. End-specific binding of DNA at the surface-exposed amino groups is obtained at optimum pH conditions. This allows a site-specific attachment and stretching of single DNA molecules at the patterned polymer film surface.

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 labeling of biomolecules has become standard practice in molecular biosciences. Modifications are used for detection, sorting and isolation of small molecules, complexes and entire cells. We have recently reported a method for introducing internal chemical and structural modifications into kbp-sized DNA target substrates that are frequently used in single-molecule experiments. It makes use of nicking enzymes that create single-stranded DNA gaps, which can be subsequently filled with labeled oligonucleotides. Here we provide a detailed protocol and further expand this method. We show that modifications can be introduced at distant loci within one molecule in a simple one-pot reaction. In addition, we achieve labeling on both strands at a specific locus, as demonstrated by Forster resonance energy transfer (FRET) experiments. The protocol requires an initial cloning of the target substrate (3–5 d), whereas the labeling itself takes 4–6 h. More elaborate purification and verification of label incorporation requires 2 h for each method.

DNA cleavage by the Type III Restriction-Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision.

A plethora of methods exist to link proteins to surfaces in order to generate functionalized materials. However, general tools that lead to functional immobilization of recombinantly expressed proteins on membranes such as liposomes or lipid-coated nanoparticles are rare. Here we present an approach that takes advantage of a double-palmitoylated peptide that mediates stable membrane anchoring in combination with protein trans-splicing for efficient immobilization of recombinant proteins fused to split intein segments. Two different DnaE split inteins from Synechocystis and Nostoc punctiforme are tested and compared to immobilization via direct native chemical ligation using a protein thioester. Protein trans-splicing proceeds at low protein concentrations and leads to functionalized vesicles and membrane-coated silica nanoparticles.

In the PLP-requiring {alpha}2{beta}2 tryptophan synthase complex, recognition of the substrate l-Ser at the {beta}-site includes a loop structure (residues {beta}110-115) extensively H-bonded to the substrate {alpha}-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: {beta}Thr110 {yields} Val, and {beta}Gln114 {yields} Asn. The {beta}T110V mutation greatly impairs both catalytic activity in the {beta}-reaction, and allosteric communication between the {alpha}- and {beta}-sites. The crystal structure of the {beta}T110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair {beta}-site catalysis and the communication of allosteric signals between the {alpha}- and {beta}-sites. Purified {beta}Q114N consists of two species of mutant protein, one with a reddish color ({lambda}max = 506 nm). The reddish species is unable to react with l-Ser. The second {beta}Q114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate l-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with l-Ser results in the formation of an inactive species during the first 15 min with {lambda}max 320 nm, followed by a slower conversion over 24 h to the species with ?max = 506 nm. The 320 and 506 nmmore » species originate from conversion of the {alpha}-aminoacrylate external aldimine to the internal aldimine and {alpha}-aminoacrylate, followed by the nucleophilic attack of {alpha}-aminoacrylate on C-4' of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4' to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of {beta}114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in tryptophan synthase.« less

The mechanism of the guanosine triphosphate (GTP) hydrolysis reaction of small G-proteins such as Ras is generally understood; however, some important molecular details are still missing. One example concerns the role of Gln61 in the catalysis of the GTP hydrolysis reaction. This amino acid is frequently mutated in oncogenic Ras leading to constitutively active variants of the protein. To elucidate the role of Gln61, subtle structural changes were introduced at this position by exchanging the natural occurring glutamine against a glutamic acid methyl ester (GluOme). Thereby the H-bond donor properties of this residue are changed and analysis of the GTP hydrolysis reaction can provide information on the function of the native carboxamide moiety. Using a semisynthetic approach, Ras(1–166)Gln61GluOMe was synthesized by sequential native chemical ligation of three unprotected peptide segments. Peptides Ras(1–50) and Ras(51–79)Gln61GluOMe were synthesized using Boc chemistry. The C-terminal peptide Ras(80–166) was expressed in E. coli. Initial tests of this semisynthetic strategy were performed by synthesis of the N- and C-terminally truncated protein variant Ras(39–101)Gln61GluOMe. The identified optimal reaction conditions were then applied to the synthesis of Ras(1–166)Gln61GluOMe. Refolding of the semisynthetic product in the presence of GTP was successful and revealed intrinsic GTPase activity of Ras(1–166)Gln61GluOMe. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 399–405, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The DnaK system from Thermus thermophilus (DnaK(Tth)) exhibits pronounced differences in organisation and regulation to its mesophile counterpart from Escherichia coli (DnaK(Eco)). While the ATPase cycle of DnaK(Eco) is tightly regulated by the concerted action of the two cofactors DnaJ(Eco) and GrpE(Eco), the DnaK(Tth) system features an imbalance in this cochaperone mediated regulation. GrpE(Tth) considerably accelerates the ATP/ADP exchange, but DnaJ(Tth) only slightly stimulates ATPase activity, believed to be a key step for chaperone activity of DnaK(Eco). By in vitro complementation assays, we could not detect significant ATPase-stimulation of orthologous DnaJ(Tth) . DnaKEco or DnaJ(Eco). DnaK(Tth)-complexes as compared to the DnaK(Eco) system, although they were nevertheless active in luciferase refolding experiments. Assistance of protein recovery by DnaK thus seems to be uncoupled of the magnitude of DnaJ mediated ATPase-stimulation.

Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.

Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding ( K d =240 nM ; Δ H=−17.6 kJ mol −1 ) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1–82) and cytoplasmic peptide (83–114) leading to an optimal and specific interaction between receptor and transducer.

Generation of biological function by chemical methods is potentially of great importance for the understanding and targeting of physiological processes. Chemical synthesis of proteins offers the ability to alter the properties of target protein molecules in a tailor-made fashion. In the present work it is demonstrated that this methodology can be expanded to the elucidation of protein–protein interactions as exemplified by the complete chemical synthesis of the protooncogene product H-Ras as well as of the Ras-binding domain (RBD) of its effector c-Raf1. The 166-aa polypeptide chain of H-Ras was synthesized by native chemical ligation of three unprotected peptide segments. Similarly, the 81-aa RBD was prepared by ligation of two peptide segments. Both RBD and Ras displayed functional and spectroscopic properties indistinguishable from their recombinant forms as judged by CD spectroscopy and from transient kinetic measurements of the Ras–RBD interaction as well as from nucleotide replacement reactions in Ras. An unnatural amino acid bearing a nitrobenzofurazan side chain was introduced into position 91 of the RBD, providing unique fluorescence properties. The association transient of nitrobenzofurazan labeled with Ras⋅guanosine 5′-β,γ-imidotriphosphate showed a slow phase that had not been detected in earlier work by using other signals.

We explore basic principles underlying the fabrication of molecularly engineered metallic nanowires through the deposition of metal onto DNA biotemplates. This novel approach takes advantage of the unique recognition, association, and binding properties of DNA, which permit both to interconnect microscopic gold electrodes with single DNA molecules in a site‐specific way and to achieve a template‐promoted growth of small metal particles along the biopolymer. We present a metallization method which suppresses kinetically, the spurious homogeneous metal nucleation, and results in a perfectly clean, in‐place metallization of biomolecules. Moreover, first experimental evidence is obtained that the metallization kinetics is strongly influenced by the composition of the DNA sequence.

Background: The Ras·GDP–Ras·GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated Ras·GTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of Ras·GTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to Ras·GTP but not to Ras·GDP. Results: The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/Ras·GTP binding surface. Additionally, a C-terminal tag consisting of His 6 was introduced. The K d values for binding of the site-specifically modified proteins to Ras·GTP are comparable to that of wild-type RBD. Immobilization of C-terminal His 6 tag-modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of Ras·GTP in the 100 nM range. Likewise, Ras·GTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. Ras·GDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. Conclusions: The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.

The nucleotide binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ, also called co-chaperones. The concerted action of the nucleotide exchange factor GrpE and the ATPase-stimulating factor DnaJ determines the ratio of the two nucleotide states of DnaK, which differ in their mode of interaction with unfolded proteins. In the Escherichia coli system, the stimulation by these two antagonists is comparable in magnitude, resulting in a balance of the two nucleotide states of DnaK(Eco) in the absence and the presence of co-chaperones. The regulation of the DnaK chaperone system from Thermus thermophilus is apparently substantially different. Here, DnaJ does not stimulate the DnaK-mediated ATP hydrolysis and thus does not appear to act as an antagonist of the nucleotide exchange factor GrpE(Tth). This raises the question of whether T. thermophilus GrpE stimulates nucleotide exchange to a smaller degree as compared to the E. coli system and how the corresponding rates relate to intrinsic ATPase and ATP binding as well as luciferase refolding kinetics of T. thermophilus DnaK. We determined dissociation constants as well as kinetic constants that describe the interactions between the T. thermophilus molecular chaperone DnaK, its nucleotide exchange factor GrpE and the fluorescent ADP analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine-5'-diphosphate by isothermal equilibrium titration calorimetry and stopped-flow kinetic experiments and investigated the influence of T. thermophilus DnaJ on the DnaK nucleotide cycle. The interaction of GrpE with the DnaK.ADP complex versus nucleotide-free DnaK can be described by a simple equilibrium system, where GrpE reduces the affinity of DnaK for ADP by a factor of about 10. Kinetic experiments indicate that the maximal acceleration of nucleotide release by GrpE is 80,000-fold at a saturating GrpE concentration. Our experiments show that in T. thermophilus, although the thermophilic DnaK system displays no stimulation of the DnaK-ATPase activity by DnaJ, nucleotide exchange is still efficiently stimulated by GrpE. This indicates that two counteracting factors are not absolutely necessary to maintain a functional and regulated chaperone cycle. This conclusion is corroborated by data that show that the slower ATPase cycle of the DnaK system as well as of heterologous T. thermophilus DnaK/E. coli DnaK systems is directly reflected in altered refolding kinetics of firefly luciferase but not necessarily in refolding yields.

The genes coding for the Thermus thermophilus (Tth) homologues of the molecular chaperones DnaK and GrpE (DnaKTth and GrpETth) were cloned and expressed in Escherichia coli. The proteins were purified and their functional properties were assessed by equilibrium and transient kinetic methods. DnaKTth has an intrinsic ATPase activity of 3x10(-4) s-1 at 25 degreesC and 10x10(-4) s-1 at 75 degreesC under single turnover conditions. It binds the fluorescent nucleotide analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-diphosphate (MABA-ADP) with a dissociation constant (Kd) of 3 nM and ADP with a Kd of 47 nM at 25 degreesC. At 75 degreesC the affinities are decreased fivefold to 15 nM (MABA-ADP) and 280 nM (ADP). The kinetic constants for two-step binding of MABA-ADP and of ADP to DnaKTth were determined at 25 degreesC and 75 degreesC, respectively. GrpETth acts as a nucleotide-exchange factor on DnaKTth and accelerates the release of bound MABA-ADP significantly. This shows that the nucleotide-binding domain is functionally intact, and that the specific interaction of DnaKTth and GrpETth is mediating nucleotide exchange.A fluorescently labelled peptide that comprises a subsequence of the E. coli transcription factor sigma32 binds to nucleotide-free DnaKTth with a Kd of 4.9 microM. Displacement with unlabelled peptide yields a Kd of 5.0 microM for the unlabelled peptide. Thus the peptide-binding domain also appears to be functional.For the cellular chaperone function of DnaK, a coupling between nucleotide and peptide-binding domains is required. However, with DnaKTth in the ATP as well as in the ADP.Pi-state, peptide is bound and released within seconds. No correlation between ATP-binding or hydrolysis by DnaKTth and changes in the sigma32 peptide exchange rates could be detected. It thus appears that the DnaK system from Th. thermophilus has a different mechanism of coupling the nucleotide state to the fast and slow peptide exchange properties.

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.

Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required.

Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools., Miniature Cas12f editing systems are well suited for in vivo editing applications. Here the authors characterize the intrinsic activity of SpCas12f1 in plant and animal cells.

Replication protein A (RPA) is involved in virtually all aspects of eukaryotic DNA processing, including replication, recombination and repair. The strong binding of RPA to single-stranded DNA (ssDNA), which provides protection from nucleolytic cleavage, is well established. Binding to partially double-stranded DNA (dsDNA) and disruption of DNA intermediates possessing secondary structures was reported previously, however the underlying mechanism remains unclear.Utilizing single-molecule magnetic tweezers, we now show that both human and yeast RPA can open a DNA hairpin subjected to force. For this well-defined substrate geometry which closely mimics a replication fork, we measured the force-dependent RPA association and dissociation, whilst resolving individual binding events.To explain the observed helix opening activity, we propose a passive model: Transient openings of the DNA helix, enhanced by stronger forces, become trapped by RPA binding. When the force is reduced, dissociation is driven by helix rehybridization. This leads to an exponential dependence of the association and dissociation rates on the applied force, in agreement with our data.By extrapolating our measured rates, we conjecture that RPA does not disrupt dsDNA on its own in the absence of force, but rather seems to rely on the active unwinding of a helicase. RPA could then strongly bind and protect ssDNA produced in its wake, providing a scaffold for the recruitment of further DNA processing enzymes. While the fork is held open, changes to the force exerted by downstream enzymes may regulate the degree of RPA binding on the ssDNA. Upon completion of the DNA processing step the fork would become unblocked. Rapid helix rehybridization then expels RPA from the ssDNA with dissociation rates on the order of hundreds of base-pairs per second, such that the completely processed dsDNA is returned to its native RPA-unbound state.