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J.G.P. Bloom

Bio: J.G.P. Bloom is an academic researcher from Delft University of Technology. The author has contributed to research in topics: Guided wave testing & Tomography. The author has an hindex of 5, co-authored 19 publications receiving 257 citations. Previous affiliations of J.G.P. Bloom include Netherlands Organisation for Applied Scientific Research.

Papers
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Journal ArticleDOI
TL;DR: By using torsionally constrained DNA molecules, the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I is reported, finding that the enzyme tracks along the helical pitch of the DNA molecule.
Abstract: Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.

115 citations

Journal ArticleDOI
TL;DR: The authors' data indicate that EcoR124I makes small steps along the DNA of 1 bp in length with 1 ATP consumed per step, but with some uncoupling of the ATPase and translocase cycles occurring so that the average number of ATP consumption per base pair slightly exceeds unity.
Abstract: The Type I restriction-modification enzyme EcoR124I is an archetypical helicase-based dsDNA translocase that moves unidirectionally along the 3′–5′ strand of intact duplex DNA. Using a combination of ensemble and single-molecule measurements, we provide estimates of two physicochemical constants that are fundamental to a full description of motor protein activity—the ATP coupling efficiency (the number of ATP consumed per base pair) and the step size (the number of base pairs transported per motor step). Our data indicate that EcoR124I makes small steps along the DNA of 1 bp in length with 1 ATP consumed per step, but with some uncoupling of the ATPase and translocase cycles occurring so that the average number of ATP consumed per base pair slightly exceeds unity. Our observations form a framework for understanding energy coupling in a great many other motors that translocate along dsDNA rather than ssDNA.

68 citations

Journal ArticleDOI
TL;DR: It is found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation, allowing dynamic control of the restriction process by the availability of motors.
Abstract: Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps—ATP binding and extrusion of an initial DNA loop—allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.

44 citations

Proceedings ArticleDOI
21 Jun 2011
TL;DR: In this article, the authors used a short spatial convolution operator to propagate a wave field through a bend to estimate the wave field at the detector ring, including the phase jump as a consequence of the natural focusing.
Abstract: The concept of predictive maintenance using permanent sensors that monitor the integrity of an installation is an interesting addition to the current method of periodic inspections. Guided wave tomography has been developed to map the wall thickness using the travel times of guided waves. The method has been demonstrated for straight pipes. The extension of this method to bends is not straightforward because natural focusing occurs due to geometrical path differences. This yields a phase jump, which complicates travel time picking. Because ray-tracing is no longer sufficient to predict the travel times a recursive wave field extrapolation has been developed. The method uses a short spatial convolution operator to propagate a wave field through a bend. The method allows to calculate the wave field at the detector ring, including the phase jump as a consequence of the natural focusing. The recursive wave field extrapolation is done in the space-frequency domain. Therefore dispersion effects can be included easily in the forward modeling. Comparison with measurements shows the accuracy of the method. The tomographic reconstruction is based on the wave field extrapolation kernel.

10 citations

01 Jan 2009
TL;DR: Guided wave travel time tomography (GWT tomography) as discussed by the authors is a promising method to monitor the wall thickness quantitatively over large areas, and it is capable of providing quantitative wall thickness information over a distance of 4 meters.
Abstract: Corrosion is one of the industries major issues regarding the integrity of assets. Currently inspections are conducted at regular intervals to ensure a sufficient integrity level of these assets. Both economical and social requirements are pushing the industry to even higher levels of availability, reliability and safety of installations. The concept of predictive maintenance using permanent sensors that monitor the integrity of an installation is an interesting addition to the current method of periodic inspections reducing uncertainty and extending inspection intervals. Guided wave travel time tomography is a promising method to monitor the wall thickness quantitatively over large areas. Obviously the robustness and reliability of such a monitoring system is of paramount importance. Laboratory experiments have been carried out on a 10″ pipe with a nominal wall thickness of 8 mm. Multiple, inline defects have been created with a realistic morphology. The depth of the defects was increased stepwise from 0.5 mm to 2 mm. Additionally the influences of the presence of liquid inside the pipe and surface roughness have been evaluated as well. Experimental results show that this method is capable of providing quantitative wall thickness information over a distance of 4 meter, with a sufficient accuracy such that results can be used for trending. The method has no problems imaging multiple defects.

9 citations


Cited by
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Journal ArticleDOI
TL;DR: A classification based on protein families that are characterized by typical sequence, structural, and mechanistic features is outlined, which complements and extends existing SF1 and SF2 helicase categorizations and highlights major structural and functional themes for these proteins.

796 citations

Journal ArticleDOI
TL;DR: New developments which provide insights into the roles of these enzymes in other aspects of cellular function are dealt with, with emphasis placed on novel hypotheses and various findings that have not yet been dealt with in a critical review.
Abstract: Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

464 citations

Journal ArticleDOI
TL;DR: Recent studies that have shed light on the still developing field of restriction-modification and on the newly re-emerging field of anti-restriction indicate that these fields continue to contribute significantly to basic science.

463 citations

Journal ArticleDOI
TL;DR: A model for generation of defined chromosomal loops is presented, based on molecular machines consisting of two coupled and oppositely directed motile elements which extrude loops from the double helix along which they translocate, while excluding one another sterically.
Abstract: The long chromosomal DNAs of cells are organized into loop domains much larger in size than individual DNA-binding enzymes, presenting the question of how formation of such structures is controlled. We present a model for generation of defined chromosomal loops, based on molecular machines consisting of two coupled and oppositely directed motile elements which extrude loops from the double helix along which they translocate, while excluding one another sterically. If these machines do not dissociate from DNA (infinite processivity), a disordered, exponential steady-state distribution of small loops is obtained. However, if dissociation and rebinding of the machines occurs at a finite rate (finite processivity), the steady state qualitatively changes to a highly ordered ‘stacked’ configuration with suppressed fluctuations, organizing a single large, stable loop domain anchored by several machines. The size of the resulting domain can be simply regulated by boundary elements, which halt the progress of the extrusion machines. Possible realizations of these types of molecular machines are discussed, with a major focus on structural maintenance of chromosome complexes and also with discussion of type I restriction enzymes. This mechanism could explain the geometrically uniform folding of eukaryote mitotic chromosomes, through extrusion of pre-programmed loops and concomitant chromosome compaction.

450 citations

Journal ArticleDOI
TL;DR: Single-molecule DNA supercoiling is used to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems and finds that Cascade has higher torque stability than Cas9 by using a conformational locking step.
Abstract: Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a site-specific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of R-loop formation has not been fully resolved. Here, we use single-molecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the R-loop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.

406 citations