Scanning Force Microscopy of DNA Translocation by the Type III Restriction Enzyme EcoP15I

Reich, Stefanie; Gössl, Illdiko; Reuter, Monika; Rabe, Jürgen P.; Krüger, Detlev H.

doi:10.1016/j.jmb.2004.06.031

Cited by 26 publications

(21 citation statements)

References 27 publications

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“…We note that whilst our results concur with other studies in bulk solution that argue against 3D DNA looping (23, 28), they are contradictory to recent AFM investigations where extensive 3D passive and active DNA looping was reported (14,16,17). However, the AFM studies could not show that DNA cleavage is directly dependent on the looped topology.…”

Section: Discussionsupporting

confidence: 66%

“…The AFM studies which support DNA looping used EcoP15I exclusively (14,16,17). EcoP15I is very closely related to EcoPI and behaves similarly (23).…”

Section: Resultsmentioning

confidence: 99%

“…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 atomic force microscope (AFM) studies (14,16,17). However, compared with Type I REs (12), Type III REs have a greatly reduced ATPase activity (4, 18), making a pure translocationdriven communication much less likely.…”

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confidence: 93%

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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.

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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Section: Discussionsupporting

confidence: 66%

“…The AFM studies which support DNA looping used EcoP15I exclusively (14,16,17). EcoP15I is very closely related to EcoPI and behaves similarly (23).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 93%

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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.

108

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“…Some models favour ATP-driven DNA translocation alone 3 or combined with three-dimensional DNA looping, 10,11 and corresponding loop structures have been visualized by atomic force microscopy and fast-scan atomic force microscopy. [10][11][12] Other researchers excluded DNA loop formation by EcoP15I and presented models that rely on one-dimensional diffusion of the enzyme along the DNA. 13,14 The diffusion is described to occur either unidirectionally first in the 5′-3′ direction from the recognition site and then switching upon reaching the DNA's 3′ end 13 or in a bidirectional random walk.…”

Section: Introductionmentioning

confidence: 99%

Functional Characterization and Modulation of the DNA Cleavage Efficiency of Type III Restriction Endonuclease EcoP15I in Its Interaction with Two Sites in the DNA Target

Möncke‐Buchner

Rothenberg

Reich

et al. 2009

Journal of Molecular Biology

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“…5 The prevailing mechanistic model to explain how EcoP15I communicates between remote recognition sites is the convergent DNA translocation of two enzymes that is predicted to produce DNA loops. 3,7,8 Reich et al 9 used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg 2+ , EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures.…”

Section: Introductionmentioning

confidence: 99%

Structural Domains in the Type III Restriction Endonuclease EcoP15I: Characterization by Limited Proteolysis, Mass Spectrometry and Insertional Mutagenesis

Wagenführ

Pieper

Mackeldanz

et al. 2007

Journal of Molecular Biology

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Scanning Force Microscopy of DNA Translocation by the Type III Restriction Enzyme EcoP15I

Cited by 26 publications

References 27 publications

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

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

Functional Characterization and Modulation of the DNA Cleavage Efficiency of Type III Restriction Endonuclease EcoP15I in Its Interaction with Two Sites in the DNA Target

Structural Domains in the Type III Restriction Endonuclease EcoP15I: Characterization by Limited Proteolysis, Mass Spectrometry and Insertional Mutagenesis

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