Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

Seidel, Ralf; Noort, John van; Scheer, Carsten van der; Bloom, Joost G P; Dekker, Nynke H.; Dutta, Christina F.; Blundell, A.J.; Robinson, Terence L.; Firman, Keith; Dekker, Cees

doi:10.1038/nsmb816

Cited by 112 publications

(168 citation statements)

References 19 publications

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“…However, it appears that EcoP15I is able to translocate against a large frictional force, which would imply an efficient coupling of the free energy of ATP hydrolysis to translocation. EcoR124I is a type I restriction enzyme also containing a superfamily 2 helicase domain; this enzyme has been shown to have a translocation velocity of 550 Ϯ 30 bp/s against applied forces of up to 5 pN (the largest force used in the experiment) (19). We have no way of extrapolating our data to low force, however it seems likely that EcoP15I will achieve a substantially increased translocation velocity in the absence of a large friction force.…”

Section: Discussionmentioning

confidence: 51%

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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

108

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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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Section: Discussionmentioning

confidence: 51%

“…It has been proposed that this barrier may be overcome by melting the DNA during translocation initiation (15), or by the formation of a (relaxed) diffusive loop before translocation, which will reduce the superhelical density (17). Translocation rates of up to 550 bp/s have been reported for type I enzymes, such as EcoR124I (18,19).…”

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

108

View full text Add to dashboard Cite

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“…3b) and can move DNA 1 or 2 bp at a time 5,57,59 . However, the current understanding of translocation by SF2 translocases involves the directional tracking (typically 3′-to-5′) of the translocase domain along the backbone of one strand of the DNA duplex (the tracking strand) 59,62,63 , and this domain would encounter a steric clash with the octamer after tracking 1 or 2 bases. A simple application of the inchworm model using this tracking mode would require a full rotation of the DNA for every ∼10 bp of translocation to maintain continuous association of the translocase domain with the tracking strand.…”

Section: The Dna-twist Conundrummentioning

confidence: 99%

Chromatin remodeling: insights and intrigue from single-molecule studies

Cairns

2007

Nat Struct Mol Biol

224

208

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Chromatin remodelers are ATP-hydrolyzing machines specialized to restructure, mobilize or eject nucleosomes, allowing regulated exposure of DNA in chromatin. Recently, remodelers have been analyzed using single-molecule techniques in real time, revealing them to be complex DNA-pumping machines. The results both support and challenge aspects of current models of remodeling, supporting the idea that the remodeler translocates or pumps DNA loops into and around the nucleosome, while also challenging earlier concepts about loop formation, the character of the loop and how it propagates. Several complex behaviors were observed, such as reverse translocation and long translocation bursts of the remodeler, without appreciable DNA twist. This review presents and discusses revised models for nucleosome sliding and ejection that integrate this new information with the earlier biochemical studies.Many processes involving chromosomes are guided by DNA-binding proteins, and the access of these proteins to DNA is regulated by chromatin. Nucleosomes, the primary repeating unit of chromatin, package DNA by wrapping 147 base pairs (bp) around an octamer of histone proteins in ∼1.7 helical turns 1-3 . This packaging provides topological order but occludes one face of the DNA, which slows or prevents the recognition of DNA sequences by DNA-binding proteins. To maintain topological order, and also to allow rapid and regulated access to the DNA, cells have evolved a set of chromatin-remodeling machines that alter nucleosome position, presence and structure.Remodelers can be separated into several families on the basis of their composition and activities: SWI/SNF, ISWI, NURD/Mi-2/CHD and the 'split ATPases' INO80 and SWR1 (which bear an insertion within their ATPase domains). Rad54 may represent an additional family, as it perturbs chromatin in vitro, but it apparently lacks specific nucleosome-interacting domains 4,5 . The participation of remodelers in various chromosomal processes has been the subject of many reviews 6-9 . The present work focuses solely on remodeler mechanisms, and primarily on the function of their conserved catalytic subunit, a superfamily 2 (SF2) ATPdependent DNA translocase. Recently, single-molecule approaches have been used to examine several remodelers, providing many new insights into their behavior. Here I discuss these recent studies, compare them with previous biochemical studies and suggest refinements to existing models to account for some of the observations. Remodelers slide, eject and restructure nucleosomesRemodelers can affect nucleosomes in at least four ways ( Fig. 1): (i) sliding, or moving the histone octamer to a new position, which exposes DNA 10-12 ; (ii) ejection, or completely displacing the octamer to expose DNA [13][14][15][16] ; (iii) removal of H2A-H2B dimers, leaving only the central H3-H4 tetramer, which exposes DNA and destabilizes the nucleosome 17,18 ; and (iv) dimer replacement-for example, exchanging the resident H2A-H2B dimers for dimers NIH Public Access

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“…The type I DNA restriction/modification enzyme EcoR124I is capable of translocating along dsDNA with an average macroscopic rate of (550 ± 30) bp/s (110). EcoR124I is also highly processive as it is capable of translocating an average of (4300 ± 900) basepairs before dissociation (110).…”

Section: Dna Translocation By Restriction Enzymesmentioning

confidence: 99%

“…The cleavage reaction begins with the recognition of the unmethylated target sequence by the complex's methyltransferase core enzyme (12,13). Initial binding is subsequently followed by ATP-dependent dsDNA translocation, performed by the two HsdR complex subunits, which pulls DNA toward the core enzyme and forming two dsDNA loops (109,110). The translocating subunits then conduct the cleavage reaction upon encountering a blockage on the DNA, including encountering a second restriction enzyme (111).…”

Section: Dna Translocation By Restriction Enzymesmentioning

confidence: 99%

All motors have to decide is what to do with the DNA that is given them

Briggs

Fischer

2014

Biomolecular Concepts

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DNA translocases are a diverse group of molecular motors responsible for a wide variety of cellular functions. The goal of this review is to identify common aspects in the mechanisms for how these enzymes couple the binding and hydrolysis of ATP to their movement along DNA. Not surprisingly, the shared structural components contained within the catalytic domains of several of these motors appear to give rise to common aspects of DNA translocation. Perhaps more interesting, however, are the differences between the families of translocases and the potential associated implications both for the functions of the members of these families and for the evolution of these families. However, as there are few translocases for which complete characterizations of the mechanisms of DNA binding, DNA translocation, and DNA-stimulated ATPase have been completed, it is difficult to form many inferences. We therefore hope that this review motivates the necessary further experimentation required for broader comparisons and conclusions.

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Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

Cited by 112 publications

References 19 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

Chromatin remodeling: insights and intrigue from single-molecule studies

All motors have to decide is what to do with the DNA that is given them

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