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To repair a DNA double-strand break by homologous recombination, 5'-terminated DNA strands must first be resected to reveal 3'-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5'-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5'-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.

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.

DNA double-strand break repair by homologous recombination employs long-range resection of the 5' DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay between them has been shown, it remains unclear whether and how these proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single-molecule level and investigated Sgs1 regulation by Dna2, the single-stranded DNA-binding protein RPA, and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection both proteins form a functional complex and couple their activities. Sgs1 drives DNA unwinding and feeds single-stranded DNA to Dna2 for degradation. RPA was found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We hypothesize that the differential regulation of Sgs1 activity by its protein partners is important to support diverse cellular functions of Sgs1 during the maintenance of genome stability.

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.

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.

Endonucleases that generate DNA double strand breaks often employ two independent subunits such that the active site from each subunit cuts either DNA strand. Restriction enzyme BcnI is a remarkable exception. It binds to the 5΄-CC/SGG-3΄ (where S = C or G, ‘/’ designates the cleavage position) target as a monomer forming an asymmetric complex, where a single catalytic center approaches the scissile phosphodiester bond in one of DNA strands. Bulk kinetic measurements have previously shown that the same BcnI molecule cuts both DNA strands at the target site without dissociation from the DNA. Here, we analyse the BcnI DNA binding and target recognition steps at the single molecule level. We find, using FRET, that BcnI adopts either ‘open’ or ‘closed’ conformation in solution. Next, we directly demonstrate that BcnI slides over long distances on DNA using 1D diffusion and show that sliding is accompanied by occasional jumping events, where the enzyme leaves the DNA and rebinds immediately at a distant site. Furthermore, we quantify the dynamics of the BcnI interactions with cognate and non-cognate DNA, and determine the preferred binding orientation of BcnI to the target site. These results provide new insights into the intricate dynamics of BcnI–DNA interactions.

Negative phototaxis in Archaea is mediated by the sensory rhodopsin II/transducer complex (NpSRII/NpHtrII). After light excitation, the signal is relayed from the receptor to NpHtrII where a rotary motion of TM2 in the membrane domain (NpHtrII1–114) is induced. This conformational change is transferred to the downstream two-component signaling cascade. Here, we describe the chemical synthesis of this membrane domain, which consists of the two transmembrane helices TM1 and TM2. NpHtrII1–114 was synthesized using two sequential ligation steps. The first ligation between NpHtrII47–59 and NpHtrII60–114 was performed in organic solvents, whereas the final ligation was successful in an aqueous buffer that contained a detergent and a denaturant. The product was refolded into micelles and showed functional properties as determined by binding studies to its cognate receptor NpSRII and by photocycle experiments. This work demonstrates that membrane proteins can be successfully synthesized by chemical means paving the way for tailor-made modifications. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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.

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

Many biological processes rely on the interaction of proteins with multiple DNA sites separated by thousands of base pairs. These long-range communication events can be driven by both the thermal motions of proteins and DNA, and directional protein motions that are rectified by ATP hydrolysis. The present review describes conflicting experiments that have sought to explain how the ATP-dependent Type III restriction–modification enzymes can cut DNA with two sites in an inverted repeat, but not DNA with two sites in direct repeat. We suggest that an ATPase activity may not automatically indicate a DNA translocase, but can alternatively indicate a molecular switch that triggers communication by thermally driven DNA sliding. The generality of this mechanism to other ATP-dependent communication processes such as mismatch repair is also discussed.

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.

SummaryCRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.

Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps—ATP binding and extrusion of an initial DNA loop—allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.

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.

Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5' flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme's capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions.

The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed., Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers. Although AtRECQ2 predominantly unwinds forked DNA substrates in a highly repetitive fashion, AtRECQ3 prefers to rewind, that is, to close preopened DNA forks. For both enzymes, this process is controlled by frequent strand switches and active sensing of the unwinding fork. The relative extent of the strand switches towards unwinding or towards rewinding determines the predominant direction of the enzyme. Our results provide a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family., RecQ helicases are enzymes that play a central role in maintaining genome stability in the DNA repair cascade. Klaue et al. show that RecQ2 and RecQ3 from Arabidopsis thaliana process DNA by, respectively, unwinding and rewinding forked DNA substrates, using a frequent strand switching mechanism.

The blue-light receptor genes (sopII) of sensory rhodopsin (SR) II were cloned from two species, the halophilic bacteria Haloarcula vallismortis (vSR-II) and Natronobacterium pharaonis (pSR-II). Upstream of both sopII gene loci, sequences corresponding to the halobacterial transducer of rhodopsin (Htr) II were recognized. In N. pharaonis, psopII and phtrII are transcribed as a single transcript. Comparison of the amino acid sequences of vHtr-II and pHtr-II with Htr-I and the chemotactic methyl-accepting proteins from Escherichia coli revealed considerable identities in the signal domain and methyl-accepting sites. Similarities with Htr-I in Halobacterium salinarium suggest a common principle in the phototaxis of extreme halophiles. Alignment of all known retinal protein sequences from Archaea identifies both SR-IIs as an additional subgroup of the family. Positions defining the retinal binding site are usually identical with the exception of Met-118 (numbering is according to the bacteriorhodopsin sequence), which might explain the typical blue color shift of SR-II to approximately 490 nm. In archaeal retinal proteins, the function can be deduced from amino acids in positions 85 and 96. Proton pumps are characterized by Asp-85 and Asp-96; chloride pumps by Thr-85 and Ala-96; and sensors by Asp-85 and Tyr-96 or Phe-96.

Cleavage of viral DNA by the bacterial Type III Restriction-Modification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication.

Helicases are widespread enzymes that share a core RecA fold and characteristic amino acid motifs which together form an ATP binding/hydrolysis site.1 For the classical helicases (i.e., those which conform to the family name), ATP-binding provides energy to drive the separation of polynucleotide duplexes into single strands. There are also many enzymes which on the basis of structure and motifs appear to be helicases, but which fulfill alternative functions without the necessity for strand separation. These include nucleoprotein remodeling, long-range motion along dsDNA and molecular signaling. The activities and mechanisms of these enzymes, often termed “helicase-like” or “pseudo-helicases,” are not well understood. Recent studies that try to elucidate the full repertoire of helicase activity provide more and more surprises for their numerous roles in genome metabolism. An example of pseudo-helicase activity is illustrated by the ATP-dependent bacterial Type III restriction endonucleases (REs). For these enzymes, site-specific recognition of a single DNA sequence is insufficient for DNA cleavage. Instead, 2 enzyme complexes that originally bind at separate target sites on the same DNA must interact to produce one double-strand break.2 Since the target sites can be many thousands of base pairs apart, the helicase domains are used to produce “long-range communication” to bring the enzymes close enough for protein–protein interaction and activation of the nuclease domain(s). According to classical helicase mechanisms,1 stepwise motion would require one ATP per base pair moved. Surprisingly, the Type III REs communicate over thousands of base pairs using only tens of ATPs.3 A number of competing models were discussed for the long-range communication of the Type III REs. In addition to dsDNA translocation and 3D DNA looping,4 ATP-independent, random 1D diffusion along the DNA (a.k.a. DNA sliding) was suggested.3 To directly reveal the communication mechanism, we developed a combined magnetic tweezers–total internal reflection fluorescence (TIRF) microscope for the direct visualization of motion by single enzyme complexes. A DNA substrate with 2 type III sites was attached at one end to a glass coverslip and at the other end to a magnetic bead, and was stretched out by the magnetic field so that it was within the evanescent excitation field. The type III RE EcoP15I was labeled with a single fluorescent quantum dot, and the protein trajectory was monitored in real time.5 We were able to observe EcoP15I binding specifically to its site, and, following a short delay, initiation of random 1D motion characteristic of DNA sliding. The diffusion coefficient for this process was one of the largest yet measured, and, given the viscous drag of enzyme and quantum dot, was consistent with uncorrelated motion with respect to the DNA helix. DNA cleavage was directly observed following collision of a sliding enzyme with a second enzyme bound at a distant site. In combination with ensemble fluorescence measurements, we could reveal that ATP hydrolysis occurred predominantly on the target site and was stringently required to initiate sliding.5 Thus, the helicase domains act as an ATP driven switch. The switching into the sliding state required about 30 ATPs (Fig. 1). A first rapid hydrolysis cycle of 10 ATPs produced a significant conformation change, which we interpret as the formation of a “sliding clamp” structure, and activation of the nuclease domain into a cleavage-competent state. Subsequent hydrolysis of 20 ATPs continued at a slower rate, until the enzyme left the site to start sliding. It is likely that the ATP hydrolysis is used to build up mechanical stress that drives the release of the enzyme from the site. The total number of ATPs required would then reflect uncoupling events, where the helicase transiently disengages from the DNA and another ATP must bind to re-establish the stressed state. Further analysis must, however, illuminate the details of the initiation process. Figure 1. Models for the ATP-dependent initiation of DNA sliding by type III restriction endonucleases5 (upper) and the MutS enzymes of mismatch repair6-8 (lower). The type III restriction enzyme EcoP15I comprises 2 Mod subunits (methyltransferase) ... Our study revealed a new functionality for helicases, namely as a molecular switch, which, in this case, initiates long-lived DNA sliding. We expect that more pseudo-helicases will be found that act as molecular switches in other processes, where latent activities need to be triggered following a recognition event. A similar sliding-based communication mechanism has now been demonstrated for the mismatch repair protein MutS, where nucleotide exchange (rather than hydrolysis) in its ABC transporter ATPase domains triggers sliding on DNA after mismatch recognition (Fig. 1).6-8 The sliding of MutS and also of its eukaryotic counterparts is used to search for strand discrimination signals on DNA that identify the damaged strand. Thus, ATP-triggered signaling through sliding is becoming a more widespread phenomenon. The difference in ATP consumption between type III REs and MutS may be due to the different ATPase classes involved. We suggest that the necessity for an ATP-triggered step to initiate sliding reflects the need for stringent control of downstream processes that in both cases results in DNA cleavage. This can be compared with the purchase of a ticket with which one is allowed to enter into the next phase. Such nucleotide-based licensing seems to provide an overall benefit to biological processes. For example, nucleotide hydrolysis is also used during translation for proofreading the binding of the correct tRNA.

To cleave DNA, Type III restriction enzymes must communicate the relative orientation of two asymmetric recognition sites over hundreds of base pairs. The basis of this long-distance communication, for which ATP hydrolysis by their helicase domains is required, is poorly understood. Several conflicting DNA-looping mechanisms have been proposed, driven either by active DNA translocation or passive 3D diffusion. Using single-molecule DNA stretching in combination with bulk-solution assays, we provide evidence that looping is both highly unlikely and unnecessary, and that communication is strictly confined to a 1D route. Integrating our results with previous data, a simple communication scheme is concluded based on 1D diffusion along DNA.

The Type I restriction-modification enzyme EcoR124I is an archetypical helicase-based dsDNA translocase that moves unidirectionally along the 3′–5′ strand of intact duplex DNA. Using a combination of ensemble and single-molecule measurements, we provide estimates of two physicochemical constants that are fundamental to a full description of motor protein activity—the ATP coupling efficiency (the number of ATP consumed per base pair) and the step size (the number of base pairs transported per motor step). Our data indicate that EcoR124I makes small steps along the DNA of 1 bp in length with 1 ATP consumed per step, but with some uncoupling of the ATPase and translocase cycles occurring so that the average number of ATP consumed per base pair slightly exceeds unity. Our observations form a framework for understanding energy coupling in a great many other motors that translocate along dsDNA rather than ssDNA.

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

Several bioinformatics studies have identified an unexpected but remarkably prevalent approximately 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.

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.

The catalytic activity and substrate channeling of the pyridoxal 5'-phosphate-dependent tryptophan synthase alpha(2)beta(2) complex is regulated by allosteric interactions that modulate the switching of the enzyme between open, low activity and closed, high activity states during the catalytic cycle. The highly conserved alphaThr183 residue is part of loop alphaL6 and is located next to the alpha-active site and forms part of the alpha-beta subunit interface. The role of the interactions of alphaThr183 in alpha-site catalysis and allosteric regulation was investigated by analyzing the kinetics and crystal structures of the isosteric mutant alphaThr183Val. The mutant displays strongly impaired allosteric alpha-beta communication, and the catalytic activity of the alpha-reaction is reduced one hundred fold, whereas the beta-activity is not affected. The structural work establishes that the basis for the missing inter-subunit signaling is the lack of loop alphaL6 closure even in the presence of the alpha-subunit ligands, 3-indolyl-D-glycerol 3'-phosphate, or 3-indolylpropanol 3'-phosphate. The structural basis for the reduced alpha-activity has its origins in the missing hydrogen bond between alphaThr183 and the catalytic residue, alphaAsp60.

ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior. The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown. We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins. In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein. In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected. These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts. It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus. This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.

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.

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).

The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.

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.

Archaeal phototaxis is mediated by sensory rhodopsins which form complexes with their cognate transducers. Whereas the receptors sensory rhodopsin I and sensory rhodopsin II (SRII) have been expressed in Escherichia coli (E. coli) only shortened fragments of HtrII from Natronomonas pharaonis (NpHtrII) are available. Here we describe the heterologous expression of full length NpHtrII which was achieved in yields of up to 0.9 mg per litre cell culture. Gel filtration analysis reveals the tendency of the transducer to form dimers and higher-order oligomers which was also observed when complexed to NpSRII. A circular dichroism (CD) spectrum of NpHtrII is comparable to those obtained for the E. coli chemoreceptors indicating a similar folding with predominantly α-helical structure. NpHtrII dissociates from the NpSRII/HtrII complex with an apparent KD of about 0.6 μM. Photocycle kinetics of the complex is comparable to that obtained for NpSRII in complex with a truncated transducer with slight differences in the M-decay. The data indicate that the heterologously expressed NpHtrII adopt a native like structure, providing the means for elucidating transmembrane signal transduction and activation of microbial signalling cascades.

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.

Helicases are ATP hydrolysis driven molecular motors that processively unwind double stranded DNA. In this study we investigate RecQ2 helicase from Arabidopsis thaliana (AtRecQ2) which plays an important role in genomic maintenance. We use high resolution magnetic tweezers in order to probe the unwinding of a DNA hairpin by this enzyme in real time under different external forces. We find an unwinding rate of 7-9 bp/s which is slow compared to many prokaryotic helicases. Applied forces between 5 and 12 pN only weakly affect this parameter, while the AT versus GC content of the unwound DNA has a significant impact. The weak force- but the relatively strong sequence dependence of DNA unwinding is in disagreement with a passive ratchet unwinding mechanism. High-resolution measurements reveal that AtRecQ2 unwinds the DNA in 3-4 bp steps. Beyond the behavior of AtRecQ2 during unwinding we analyze its behavior on single-strand DNA. While failing to detect single-strand DNA translocation, the data in contrary suggests that AtRecQ2 diffuses on single stranded DNA. Such a weak contact to single-strand DNA is supported by the observation that even on a stretched hairpin configuration the enzyme is capable of repetitive shuffling, i.e. the instantaneous restart of DNA unwinding after a terminated event. Based on this we hypothesize that AtRecQ2 switches between a processive unwinding-mode on double-stranded DNA and a diffusive-mode on single-stranded DNA in order to keep a target hairpin constantly open or to search for a new distant target fork.

During transcription RNA polymerase frequently evicts the H2A-H2B dimers from nucleosomes. This might be necessary to access the DNA but is also believed to mediate a feedback mechanism by which transcription of a gene facilitates the exchange of epigenetic marks and histone variants. This in turn can then immediately influence gene expression by consecutive RNA polymerases. Little is known about the role that H2A-H2B dimers play in the response of chromatin to mechanical stresses during transcription.In order to elucidate the fate of dimers during transcription, we studied DNA unwrapping from single nucleosomes in the presence of an external force using magnetic tweezers. We subjected reconstituted nucleosomal arrays, containing up to 48 nucleosomes, to tension and monitored the DNA length in relation to force. We find significant differences in the DNA unwrapping forces between nucleosomes containing canonical histones only, the H2AvD histone variant and H3-H4 tetramers. This reveals an important role of the H2A-H2B dimer during mechanical DNA disruption from the nucleosome, as might occur during transcription.