Clustered, regularly interspaced, short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a sitespecific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of Rloop formation has not been fully resolved. Here, we use singlemolecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade-and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the Rloop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.magnetic tweezers | genome engineering | crRNA C lustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems constitute an adaptable immune system that protects bacteria and archaea against foreign nucleic acids. The defense is initiated by a ribonucleoprotein (RNP) complex that mediates cleavage of dsDNA (1) or RNA (2, 3). The RNA component (crRNA) of the complex is derived by transcription and posttranscriptional processing from a locus containing CRISPRs (2, 4, 5) in which short spacer fragments were integrated from foreign nucleic acids (6-8). Each transcribed crRNA spacer sequence encodes the recognition of the targets. In DNA-targeting CRISPR-Cas systems, the crRNAs form a hybrid with a matching complement (protospacer) on an invading DNA, which leads to the displacement of the noncomplementary strand. The resulting structure is called an R-loop and constitutes the signal for subsequent DNA degradation. R-loop formation is additionally dependent on a short protospacer adjacent motif (PAM) (Fig. 1A), which provides discrimination between self and nonself DNA in CRISPR systems; it is absolutely required for recognition of the invading DNA but is absent from the host CRISPR array (9).On the basis of sequence homology, different CRISPR-Cas families have been identified (10). We investigate here a type IE and a type II system from Streptococcus thermophilus St-CRISPR4 and St-CRISPR3, respectively. The Cas proteins of type IE systems (4, 11, 12) associate with a crRNA into a mult...

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 speci®cally 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 intersite distance gives a translocation velocity of 400 6 32 bp/s at 20°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 caǹ reset' in either direction whenever the DNA is released.

Helicases are ubiquitous ATPases with widespread roles in genome metabolism. Here we report a new functionality for ATPases with helicase-like domains, namely that ATP hydrolysis can trigger ATP-independent long-range protein diffusion on DNA in one dimension (1D). Specifically, using single-molecule fluorescence microscopy we show that the Type III restriction enzyme EcoP15I uses its ATPase to switch into a distinct structural state that diffuses on DNA over long distances and long times. The switching occurs only upon binding to the target site and requires hydrolysis of ~30 ATPs. This study defines the mechanism for these enzymes and shows how ATPase activity is involved in DNA target site verification and 1D signaling, roles that are common in DNA metabolism, for example, in nucleotide excision and mismatch repair.Helicases were defined classically as ATPases that use directional translocation to unwind duplex nucleic acids. More recently however, many "pseudo-helicases" have been described that posses amino acid motifs characteristic of helicases yet fulfill their cellular functions without obligate DNA unwinding (1) and instead are often translocases that move directionally on single-(2) or double-stranded (3,4) nucleic acids. Pseudo-helicases include the ATP-dependent bacterial Type I and III restriction enzymes (REs) which use ATP hydrolysis to communicate between two distant restriction sites on the same DNA (5,6). Only if both sites are unmethylated is the DNA considered "foreign" and destroyed by the endonuclease. Type III REs form a heterooligomeric complex of two subunits: Mod (for target site methylation) and Res (for ATPase and endonuclease activities). Because they only hemimethylate their asymmetric recognition sequences, these enzymes must signal the relative site orientation during communication (7): cleavage occurs only if the sites are in an inverted-repeat orientation, arranged either "head-to-head" (HtH) (7) or "tail-to-tail" (TtT) (8). Type III REs hydrolyze only a few tens of ATP per dsDNA break (9,10), suggesting either passive three-dimensional looping between sites with limited translocation (11) or diffusion in 1D following an ATP-dependent switch (Fig. S1A) To distinguish between the different communication models, we developed a real-time single-molecule assay using magnetic tweezers combined with TIRF microscopy (12) that can localize fluorescently-labeled enzymes on stretched 26 kbp DNA molecules containing two centrally-located HtH EcoP15I sites with 6 kbp intersite distance (Fig. 1A). EcoP15I was labeled with quantum dots on the C-terminus of the Res subunit (QR-EcoP15I), without compromising the biochemical activity (Fig. S2). While DNA binding by lone quantum dots was not detected, addition of QR-EcoP15I and ATP resulted in enzyme attachment at one ( Fig. 1B and movie S1) or two ( Fig. S3) specific recognition sites. Enzymes always arrived instantaneously at the sites, with no evidence for long-lived non-specific binding en route (N = 61). Following a delay of, on av...

Most reactions on DNA are carried out by multimeric protein complexes that interact with two or more sites in the DNA and thus loop out the DNA between the sites. The enzymes that catalyze these reactions usually have no activity until they interact with both sites. This review examines the mechanisms for the assembly of protein complexes spanning two DNA sites and the resultant triggering of enzyme activity. There are two main routes for bringing together distant DNA sites in an enzyme complex: either the proteins bind concurrently to both sites and capture the intervening DNA in a loop, or they translocate the DNA between one site and another into an expanding loop, by an energy-dependent translocation mechanism. Both capture and translocation mechanisms are discussed here, with reference to the various types of restriction endonuclease that interact with two recognition sites before cleaving DNA.

The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5-or 10-bp gaps in the 3-5 strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3-5-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.A general feature of eukaryotes is that their genomic DNA associates with proteins to form chromatin. In addition to providing a means of packaging DNA within nuclei, chromatin provides an additional level at which gene expression can be regulated. Active regions of the genome tend to be maintained in a more accessible state than inactive regions, and the manipulation of chromatin structure has been found to play an important role in the regulation of many genes. In order to be able to regulate gene expression at this level, eukaryotes have devised a number of strategies by which they can manipulate chromatin structure. These include the covalent modification of chromatin structure by posttranslational modification, the manipulation of the protein content of chromatin through the association of variant histone and nonhistone proteins, and the noncovalent alteration of chromatin structure by ATP-dependent chromatin-remodeling activities (2,7,38,67).It now appears likely that all eukaryotes encode multiple yet distinct ATPases with homology to the yeast SNF2 protein (Snf2p). These include the Sth1p, Chd1p, Isw1p, Isw2p, and Ino80p proteins in budding yeast and a spectrum of related proteins in higher eukaryotes (2, 66). Among these, the ISWI proteins represent a discrete class of ATPase involved in chromatin remodeling. The founding member of the ISWI group was identified in Drosophila melanogaster due to its similarity to Snf2p. As the homology is restricted to the helicase-like domain, it was named imitation SWI/SNF (ISWI) (21). Biochemical characterization of ISWI proteins began with the identification of ISWI as the catalytic subunit of three distinct complexes, nucleosome-remodeling factor (NuRF), chromatin accessibility complex (ChrAC), and the ATP-utilizing chromatin assembly and modifying factor (ACF) isolated from Drosophila embryo extracts (36,60,64). Subsequently, ISWI-related proteins have been found to be components of chromatin-remodeling complexes in organisms ranging from yeast to humans, suggesting that these proteins fulfill important functions conserved throughout evolution (5,42,43,49,61). Within each of these complexes, the ISWI protein is associated with a...