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

Abstract

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 ...

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

“…We found that the ATPase rates were, within experimental error, similar on the HtH and TtT substrates and were independent of endcapping. As found previously (30)…”

“…For both enzymes and both HtH and TtT orientations, the single-molecule and bulk solution data coincide, within experimental error. Given the different DNA configurations in the tweezers (DNA stretched by applied force) and in bulk solution (no applied force and DNA as a random coil), the similar kinetics provide further evidence for force-independent one-dimensional communication, as noted previously for HtH DNA using a broad range of forces (30). In comparison, neither of the two enzymes produce any significant cleavage of HtT DNA; the comparable data from ref.…”

“…In comparison, neither of the two enzymes produce any significant cleavage of HtT DNA; the comparable data from ref. 30 in Fig. 2B shows cleavage confined to <10% of molecules and with a drastically slower rate.…”

“…Recently we have shown that cleavage efficiency of linear HtH DNA can be dramatically restored by binding physical roadblocks, such as streptavidin, at the DNA ends, which we term "end-capping" (30). In contrast, the poor cleavage activity on substrates with sites in direct (HtT) repeat was unaffected by end-capping.…”

“…Additional embellishments to the model including one-dimensional translocation without looping (28) and additional three-dimensional diffusive looping (29), have also been suggested. More recently, a single-molecule study provided unambiguous evidence that both passive and active DNA looping are not on-pathway to cleavage and that communication occurs bidirectionally in onedimension along the DNA contour (30). Together with the low ATPase rates of the Type III enzymes (1,000-fold lower than for Type I enzymes, 22,23,30), which realistically rules out substantial motor-driven movement, a new model was developed in which motion along DNA occurs by thermally-driven onedimensional diffusion, i.e., DNA sliding (20 and 30).…”