When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I

Stanley, Louise K.; Seidel, Ralf; Scheer, Carsten van der; Dekker, Nynke H.; Szczelkun, Mark D.; Dekker, Cees

doi:10.1038/sj.emboj.7601104

Cited by 61 publications

(89 citation statements)

References 44 publications

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“…The unwinding of the DNA is inherent to the function of helicases such as RNA polymerase (9), the mismatch repair MutS proteins (10) and HSV-1 UL9 protein (11,12), and hence these helicases must track the DNA. However, in the case of restriction enzymes (13) and the chromatin remodelling Snf2 proteins (14), there is no functional requirement to unwind the DNA. For instance, EcoR124I, a type I restriction enzyme and a superfamily 2 helicase, has been shown to track the DNA double helix without substantial strand separation.…”

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confidence: 99%

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

Crampton

Yokokawa

Dryden

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

115

111

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Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1-3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 ؎ 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPasedriven translocation of the intervening DNA contour.imaging ͉ nucleic acid ͉ restriction-modification enzyme ͉ scanning-probe

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mentioning

confidence: 99%

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

Crampton

Yokokawa

Dryden

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

115

111

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“…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).…”

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confidence: 99%

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

Ramanathan

Aelst

Sears

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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108

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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 ...

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“…DNA translocation involves a motor activity, which has led to these enzymes being included in the SFII superfamily of closely related DNA helicases (22). However, this motion is not due to a simple helicase activity (83).…”

Section: Background Of R-m Systemsmentioning

confidence: 99%

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

Youell

Firman

2008

Microbiol Mol Biol Rev

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SUMMARY Plasmid R124 was first described in 1972 as being a new member of incompatibility group IncFIV, yet early physical investigations of plasmid DNA showed that this type of classification was more complex than first imagined. Throughout the history of the study of this plasmid, there have been many unexpected observations. Therefore, in this review, we describe the history of our understanding of this plasmid and the type I restriction-modification (R-M) system that it encodes, which will allow an opportunity to correct errors, or misunderstandings, that have arisen in the literature. We also describe the characterization of the R-M enzyme EcoR124I and describe the unusual properties of both type I R-M enzymes and EcoR124I in particular. As we approached the 21st century, we began to see the potential of the EcoR124I R-M enzyme as a useful molecular motor, and this leads to a description of recent work that has shown that the R-M enzyme can be used as a nanoactuator. Therefore, this is a history that takes us from a plasmid isolated from (presumably) an infected source to the potential use of the plasmid-encoded R-M enzyme in bionanotechnology.

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When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I

Cited by 61 publications

References 44 publications

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

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

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

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