5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage.

A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.

The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a.

Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with Streptococcus thermophilus DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.

Structural insights into DNA sequence recognition by Type ISP restriction-modification enzymes.

Engineering restriction enzymes with new sequence specificity has been an unaccomplished challenge, presumably because of the complexity of target recognition. Here we report detailed analyses of target recognition by Type ISP restriction-modification enzymes. We determined the structure of the Type ISP enzyme LlaGI bound to its target and compared it with the previously reported structure of a close homologue that binds to a distinct target, LlaBIII. The comparison revealed that, although the two enzymes use almost a similar set of structural elements for target recognition, the residues that read the bases vary. Change in specificity resulted not only from appropriate substitution of amino acids that contacted the bases but also from new contacts made by positionally distinct residues directly or through a water bridge. Sequence analyses of 552 Type ISP enzymes showed that the structural elements involved in target recognition of LlaGI and LlaBIII were structurally well-conserved but sequentially less-conserved. In addition, the residue positions within these structural elements were under strong evolutionary constraint, highlighting the functional importance of these regions. The comparative study helped decipher a partial consensus code for target recognition by Type ISP enzymes.

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp.

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes.

Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission.

The helicase-like domains of type III restriction enzymes trigger long-range diffusion along DNA.

Helicases are ubiquitous adenosine triphosphatases (ATPases) with widespread roles in genome metabolism. Here, we report a previously undescribed 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. We define the mechanism for these enzymes and show 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.

DNA cleavage by Type ISP Restriction-Modification enzymes is initially targeted to the 3'-5' strand.

The mechanism by which a double-stranded DNA break is produced following collision of two translocating Type I Restriction-Modification enzymes is not fully understood. Here, we demonstrate that the related Type ISP Restriction-Modification enzymes LlaGI and LlaBIII can cooperate to cleave DNA following convergent translocation and collision. When one of these enzymes is a mutant protein that lacks endonuclease activity, DNA cleavage of the 3'-5' strand relative to the wild-type enzyme still occurs, with the same kinetics and at the same collision loci as for a reaction between two wild-type enzymes. The DNA nicking activity of the wild-type enzyme is still activated by a protein variant entirely lacking the Mrr nuclease domain and by a helicase mutant that cannot translocate. However, the helicase mutant cannot cleave the DNA despite the presence of an intact nuclease domain. Cleavage by the wild-type enzyme is not activated by unrelated protein roadblocks. We suggest that the nuclease activity of the Type ISP enzymes is activated following collision with another Type ISP enzyme and requires adenosine triphosphate binding/hydrolysis but, surprisingly, does not require interaction between the nuclease domains. Following the initial rapid endonuclease activity, additional DNA cleavage events then occur more slowly, leading to further processing of the initial double-stranded DNA break.

The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites.

The Type ISP Restriction-Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5'-TnAGCC-3', where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K(+) ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation-collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K(+) ions produces a far more distributive cleavage profile.

Dissociation from DNA of Type III Restriction-Modification enzymes during helicase-dependent motion and following endonuclease activity.

DNA cleavage by the Type III Restriction-Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼ 200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can 'turnover', albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. 'DNA sliding').

DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion.

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.

Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease.

AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat.

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.

Type III restriction enzymes communicate in 1D without looping between their target sites.

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.