Single-Molecule Insight Into Target Recognition by CRISPR-Cas Complexes.

Ribonucleoprotein (RNP) complexes from CRISPR-Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR-Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule. Specifically, we demonstrate how magnetic tweezers are applied to measure the local DNA unwinding at the target in real time. To this end we introduce the technique and the measurement principle. By studying modifications of the consensus target sequence, we show how different sequence elements contribute to the target recognition mechanism. From these data, a unified target recognition mechanism can be concluded for the RNPs Cascade and Cas9 from types I and II CRISPR-Cas systems. R-loop formation is hereby initiated on the target at an upstream element, called protospacer adjacent motif (PAM), from which the R-loop structure zips directionally toward the PAM-distal end of the target. At mismatch positions, the R-loop propagation stalls and further propagation competes with collapse of the structure. Upon full R-loop zipping conformational changes within the RNPs trigger degradation of the DNA target. This represents a shared labor mechanism in which zipping between nucleic acid strands is the actual target recognition mechanism while sensing of the R-loop arrival at the PAM-distal end just verifies the success of the full zipping.

A new detection system for extremely small vertically mounted cantilevers.

Detection techniques currently used in scanning force microscopy impose limitations on the geometrical dimensions of the probes and, as a consequence, on their force sensitivity and temporal response. A new technique, based on scattered evanescent electromagnetic waves (SEW), is presented here that can detect the displacement of the extreme end of a vertically mounted cantilever. The resolution of this method is tested using different cantilever sizes and a theoretical model is developed to maximize the detection sensitivity. The applications presented here clearly show that the SEW detection system enables the use of force sensors with sub-micron size, opening new possibilities in the investigation of biomolecular systems and high speed imaging. Two types of cantilevers were successfully tested: a high force sensitivity lever with a spring constant of 0.17 pN nm(-1) and a resonant frequency of 32 kHz; and a high speed lever with a spring constant of 50 pN nm(-1) and a resonant frequency of 1.8 MHz. Both these force sensors were fabricated by modifying commercial microcantilevers in a focused ion beam system. It is important to emphasize that these modified cantilevers could not be detected by the conventional optical detection system used in commercial atomic force microscopes.

Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites.

Before cleaving DNA substrates with two recognition sites, the Cfr10I, NgoMIV, NaeI and SfiI restriction endonucleases bridge the two sites through 3D space, looping out the intervening DNA. To characterise their looping interactions, the enzymes were added to plasmids with two recognition sites interspersed with two res sites for site-specific recombination by Tn21 resolvase, in buffers that contained either EDTA or CaCl2 so as to preclude DNA cleavage by the endonuclease; the extent to which the res sites were sequestered into separate loops was evaluated from the degree of inhibition of resolvase. With Cfr10I, a looped complex was detected in the presence but not in the absence of Ca(2+); it had a lifetime of about 90 seconds. Neither NgoMIV nor NaeI gave looped complexes of sufficient stability to be detected by this method. In contrast, SfiI with Ca(2+) produced a looped complex that survived for more than seven hours, whereas its looping interaction in EDTA lasts for about four minutes. When resolvase was added to a SfiI binding reaction in EDTA followed immediately by CaCl2, the looped DNA was blocked from recombination while the unlooped DNA underwent recombination. By measuring the distribution between looped and unlooped DNA at various SfiI concentrations, and by fitting the data to a model for DNA binding by a tetrameric protein to two sites in cis, an equilibrium constant for the looping interaction was determined. The equilibrium constant was essentially independent of the length of DNA between the SfiI sites.

Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I.

DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterized the structure and mode of action of the related EcoP1I and EcoP15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res(2)Mod(2) subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circular substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP1I resulted in cutting the bottom DNA strand at the EcoP15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.

One- and three-dimensional pathways for proteins to reach specific DNA sites.

Proteins that interact with specific DNA sites bind to DNA at random and then translocate to the target site. This may occur by one-dimensional diffusion along the DNA, or through three-dimensional space via multiple dissociation/re-associations. To distinguish these routes, reactions of the ECO:RV endonuclease were studied on substrates with two ECO:RV sites separated by varied distances. The fraction of encounters between the DNA and the protein that resulted in the processive cleavage of both sites decreased as the length of intervening DNA was increased, but not in the manner demanded for one-dimensional diffusion. The variation in processivity with inter-site spacing shows instead that protein moves from one site to another through three-dimensional space, by successive dissociation/re-associations, though each re-association to a new site is followed by a search of the DNA immediately adjacent to that site. Although DNA-binding proteins are usually thought to find their target sites by one-dimensional pathways, three-dimensional routes may be more common than previously anticipated.

Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement.

The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein-directed displacement of a DNA triplex, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease. Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics. As the distance between type I and triplex sites was shortened, the lag time decreased whilst the displacement reaction remained exponential. This is indicative of processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400+/-32 bp/s at 20 degrees C. Furthermore, the data can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can 'reset' in either direction whenever the DNA is released.

How to proteins move along DNA? Lessons from type-I and type-III restriction endonucleases.

Protein-mediated communications on DNA are universally important. The translocation of DNA driven by a high-energy phosphoryl potential allows long stretches of DNA to be traversed without dissociation. Type-I and type-III enzymes both use a common DNA-tracking mechanism to move along DNA, dependent on the hydrolysis of ATP. Type-I enzymes cleave DNA at distant DNA sites (and in some cases close to the site), due to a stall in enzyme motion. This can be due to collision with another translocating type-I enzyme or, on circular DNA, due to an increased topological load. ATP hydrolysis is considerable, and continues after DNA cleavage. Type-III enzymes only cleave DNA proximal to their sites due to collision between two endonucleases tracking with defined polarity. ATP hydrolysis is less than with the type-I enzymes. Homology to DNA helicases has been found within the HsdR and Res subunits. Mutagenesis of the DEAD-box motifs affects both ATP hydrolysis and DNA cleavage. This demonstrates a tight link between ATPase and endonuclease activities. A strand-separation mechanism akin to the DNA helicases is a possibility. The DNA-based motor proteins are mechanistically ill-defined. Further study using some of the techniques pioneered with classical motor proteins will be needed to reveal more detail.

DNA excision by the Sfi I restriction endonuclease.

A mechanism for the precise excision of DNA between two target sites was elucidated by analysing the individual steps during the reactions of the SfiI endonuclease on a plasmid with two SfiI sites. Previous studies had indicated that SfiI is a tetrameric protein that binds to two copies of its recognition site before cleaving both sites in both strands. In this study, the concerted cleavage of four phosphodiester bonds was shown to arise from four consecutive reactions that had similar values for their intrinsic rate constants. Each reaction is presumably mediated by one of the four active sites in the tetramer and all four were generally completed within the life-time of the complex between the protein and two recognition sites, though products cleaved in one or two phosphodiester bonds were also detected following premature dissociation of the enzyme-substrate complex at elevated temperatures. At the physiological temperature for this enzyme, all four bonds were cleaved within one minute but the subsequent dissociation of the enzyme-product complex, liberating the excised segment of DNA, took about one hour. The tetrameric structure for SfiI was confirmed by equilibrium centrifugation.

Selection of non-specific DNA cleavage sites by the type IC restriction endonuclease EcoR124I.

The Type IC restriction endonuclease EcoR124I binds specifically to its recognition sequence but subsequently translocates non-specific DNA past the complex in an ATP-dependent mechanism. The enzyme thus has the potential to cleave DNA at loci distant from the recognition site. We have scrutinised the link between translocation and cleavage on linear and circular DNA substrates. On linear DNA carrying two recognition sites, the majority of cleavages at loci distant from the recognition site occurred between the two sites, regardless of the inter-site distance or relative orientations. On circular DNA carrying one site, distant cleavages occurred throughout the DNA but an equivalent linear molecule underwent considerably fewer cleavages at distant loci. These results agree with published models for DNA tracking. However, on every molecule investigated, discrete cleavage sites were also observed within +/-250 bp of the recognition sites. The localised cleavages were not confined to particular DNA sequences and were independent of DNA topology. We propose a model to account for both distant and localised cleavage events. The conformation of the DNA loop extruded during tracking may result in two DNA segments being held in proximity to the restriction moiety on the protein, one close to the EcoR124I site and another distant from the site: cleavage may occur in either segment. Alternatively, the cutting of DNA close to recognition sites may be the result of multiple nicks being generated in the expanding loop before any extensive translocation.

Random walk models for DNA synapsis by resolvase.

During site-specific recombination by resolvase, the protein binds to two sites on a supercoiled DNA molecule and the loaded sites then interact with each other to form a synaptic complex. The kinetics of synapsis show non-exponential behaviour extending over five log units of time and are independent of the length of the DNA molecule and the length of DNA between the sites. In this study, numerical models were developed in order to account for how fluctuations in the structure of supercoiled DNA might lead to the juxtaposition of distant sites in a manner consistent with the experimental data on synapsis by resolvase. Models where the juxtaposition arises from fluctuations around branch points in the superhelix failed to match the data: they yielded non-exponential kinetics but only over two log units of time and they predicted longer synapsis times for both larger DNA molecules and larger inter-site spacings. In another model, one fraction of the juxtaposition events gives rise directly to the productive complex while the remaining fraction initially yields a non-productive complex: the latter molecules undergo no further fluctuations until the abortive synapse dissociates at the end of a delay period. This model again failed to match the experimental data. However, the inclusion of three sorts of non-productive complexes, each with a different delay constant, led to progress curves that concurred with the data. Schemes were also developed to account for the juxtaposition of three sites at a branch point in supercoiled DNA.

Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates.

Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.

Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease.

The SfiI endonuclease differs from other type II restriction enzymes by cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The nature of this communication event between distant DNA sites was analysed on plasmids with recognition sites for SfiI interspersed with recombination sites for resolvase. These were converted by resolvase to catenanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions between SfiI sites on the same DNA therefore cannot follow the DNA contour and, instead, must stem from their physical proximity. In buffer lacking Mg2+, where SfiI is inactive while resolvase is active, the addition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging its sites and thus isolating the sites for resolvase into separate loops. The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+.

A general assay for restriction endonucleases and other DNA-modifying enzymes with plasmid substrates.

A procedure for measuring the activities of enzymes that alter the covalent structure of DNA is described. The assay utilizes covalently closed circles of DNA as the substrate and yields quantitative data on the fraction of this DNA converted to both open-circle and linear forms.

Recombination. Pieces of the site-specific recombination puzzle.

Understanding how nucleoprotein complexes interact with specific DNA sequences has come closer with structural and mechanistic studies of the interaction between the recombination enzyme resolvase and DNA.

Probing the protein-DNA interface of the EcoRV modification methyltransferase bound to its recognition sequence, GATATC.

The DNA contacts produced between the EcoRV modification methyltransferase and its recognition sequence, GATATC, have been determined. The enzyme's general location in a methylase/DNA/sinefungin ternary complex was evaluated by protection from exonuclease III digestion. Important phosphate contacts were resolved using N-ethyl-N-nitrosourea ethylation interference footprinting. Methylation protection and interference using dimethyl sulfate were employed to assess significant contacts to purinic bases. The protein-DNA interface was further probed using oligodeoxynucleotides containing base analogues within the GATATC sequence. Most of the experiments were carried out using hemimethylated sequences, i.e., having 6-methyladenosine at the methylation site in one of the strands. The monomeric methylase was found to bind to the DNA in two different orientations for the methylation of each strand. The enzyme approaches the DNA, predominantly from one "side", and makes most of its contacts in the major groove. In either of the two binding events contacts are made to the four phosphates NpNpNpGpA and the three bases GAT (where GAT represents the 5' half of the GATATC site) on both DNA strands. The phosphates and bases in the 3' ATC half are much less important. Although the contacts made to the equivalent locations on each strand are similar, they display a slight but consistent change dependent on which strand contains the 6-methyldeoxyadenosine. This strand variation shows completely reciprocal behavior, switching around exactly, depending entirely on the methylated deoxyadenosine location. It is this that provides evidence for the two binding modes. The results obtained are discussed in terms of possible models for the protein-DNA interface.

Sequence-specific binding of DNA by the EcoRV restriction and modification enzymes with nucleic acid and cofactor analogues.

The DNA-binding properties of the EcoRV restriction endonuclease and modification methyltransferase with their recognition sequence (GATATC) were analyzed using the electrophoretic band-shift assay. It has previously been observed that the endonuclease does not bind specifically to GATATC sequences in the absence of the essential cofactor Mg2+. To investigate any possible roles for Mg2+ in promoting specific DNA binding, a set of hydrolysis-resistant oligonucleotide substrates were synthesized that contained either phosphate (phosphorothioate, 3'-S-phosphorothiolate), sugar (4'-thiothymidine), or base (7-deaza-2'-deoxyadenosine) modifications. However, it was found that none of these were specifically bound by the endonuclease in either the absence or the presence of Mg2+. In contrast, the methylase bound to GATATC sequences much more strongly than to nonspecific sites, and it was possible to observe the formation of enzyme--DNA complexes by gel retardation. Binding to GATATC sequences was increased by the addition of sinefungin, a nonreactive analogue of the essential cofactor S-adenosyl-L-methionine (AdoMet). Presumably this also occurs with AdoMet although methylation and turnover prevented its direct observation. In the presence of sinefungin the strongest binding was observed with hemimethylated EcoRV sequences (Kd = 11-13 nM), and unmethylated DNA was bound less well (Kd = 46 nM). Specific, albeit weaker binding was also seen with the dimethylated product (Kd = 143 nM). A difference in electrophoretic mobility was observed between enzyme-substrate and enzyme-product complexes suggestive of structural differences between them. The Kapp value found for sinefungin, with the hemimethylated EcoRV sequence, was 10.9 mM.