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...

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.T he ability of enzymes bound at distant DNA sites to communicate with each other via long-range interactions is an important biological theme. Very often the underlying genetic processes, such as gene silencing, site-specific recombination, restriction, etc., rely on energy-independent DNA looping (1). For many other processes, such as in mismatch repair (2) and for both Type I and III restriction enzymes (REs) (3, 4), the long-range interaction relies on ATP hydrolysis and in these cases the contribution of general passive three-dimensional (3D) looping to communication remains controversial.Restriction enzymes are a model family for studying longrange communication because the majority (and in particular all Type I and III REs) need to interact with two separate DNA sequences before cutting DNA. For the Type II REs, there is growing evidence that passive 3D DNA looping (Fig. 1A) is frequently used (5). In contrast, the Type I and III REs contain protein domains that are classified as Superfamily 2 (SF2) DNA helicases (6), and these domains are required for ATPdependent DNA communication (7,8). The role of the helicase domains in the Type I REs has been clearly defined ( Fig. 1 A); communication involves DNA loop extrusion driven by directional dsDNA translocation (9, 10), without DNA unwinding (11), with the motor making steps along the DNA of Ͻ2 bp and consuming on average one ATP for each bp moved (12). Cleavage occurs upon collision with a second translocating motor at random positions distant from the binding site (3). This is therefore a pure 1D directional communication process. In comparison, the communication mechanism for Type III REs has not been accurately defined and conflicting models have been proposed (4,13,14).Type III REs require two copies of their asymmetric recognition site in an indirectly repeated, head-to-head (HtH) orientation (15) (Fig. 1 A) cleaving the DNA 25-27 bp downstream of only one of the two sites. Given that Type III REs also require the ATPase activity of their SF2 helicase domains [albeit unrelated to Type I REs (6)], a Type I-like DNA loop translocation model was proposed in which translocation was unidirectional, accounting for the site-orientation preference (4). In support of this model, apparent DNA looping activity has been observed in ...

Cleavage of viral DNA by the bacterial Type III RestrictionModification 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.diffusion | helicase | motor | switch A lmost every genetic process requires protein complexes to interact simultaneously with specific DNA sites or structures that are located at-a-distance along the genome, including events in DNA replication, repair, recombination, and transcription. In many cases these so-called "long-range communications" are independent of the relative orientation of the interacting sites on the DNA (1). E.g., gene activation by remote enhancer elements is associated with random DNA looping between regulatory elements, i.e., chromatin loop formation (2). Such reactions can occur on DNA of any topology, and can even occur between sites on separate DNA molecules providing the local concentration is sufficiently elevated (1). In other cases however, a successful interaction only occurs when the sites are located on the same DNA in a specific relative orientation. The reaction can then be said to have "site-orientation selectivity," which can be achieved using both NTP-independent or NTP-dependent pathways:Site-specific recombinases (SSRs) have provided a mechanistic framework for NTP-independent communication (3-5). For example, the transposon-encoded resolvases have a strong preference to recombine sites in direct repeat on the same DNA (6). Site-orientation selectivity is important as uncontrolled rearrangements of DNA sequences may result in loss of function. For all SSRs studied to-date, the long-range communication occurs by the sites interacting via thermally driven three-dimensional diffusion, i.e., DNA looping (7). However, to achieve site-orientation selectivity, the geometry of the resulting site-site synapse is biased by an energetic "filter" that can be a preference for DNA substrates with a particular topology (e.g., 8) or the requirement for accessory DNA-binding factors (e.g., 9). For example, recombination by the resolvases requires the capture of three DNA nodes which are significantly favored by negative supercoiling (6,8,(10)(11)(12).For many processes that are NTP-dependent, site-orientation selectivity comes from directional one-dimensional motion along DNA. A classical example is transcription-...

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, 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 where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission.

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.