Site-Specific DNA-nicking Mutants of the Heterodimeric Restriction Endonuclease R.BbvCI

Heiter, Daniel F.; Lunnen, Keith D.; Wilson, Geoffrey G.

doi:10.1016/j.jmb.2005.02.034

Cited by 81 publications

(90 citation statements)

References 39 publications

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“…The strategy developed here, to determine whether a protein maintains contact with the DNA as it moves from one site to another, requires a protein that interacts differently with each strand of the DNA. The BbvCI restriction enzyme meets this requirement, as a result of two of its properties (30). First, it recognizes a nonpalindromic site with different 5Ј-3Ј sequences in each strand…”

Section: Resultsmentioning

confidence: 99%

“…The subunits are each specific for an individual strand: R 1 cuts the GC strand and R 2 cuts the CC strand (30). Certain mutations in the R 1 subunit leave R 1 Ϫ R 2 ϩ proteins that cut only the CC strand; conversely, R 1 ϩ R 2 Ϫ proteins cut only the GC strand.…”

Section: Resultsmentioning

confidence: 99%

“…First, the assays described below measure products with double-strand breaks, and the rate at which wild-type BbvCI cuts both strands is limited by R 2 on the CC strand, not by R 1 on the GC strand (31). Second, some experiments were done with DNA that had been nicked at both sites in the GC sequence by using the R 1 ϩ R 2 Ϫ mutant of BbvCI (30). The nicked DNA was tested with wild-type BbvCI, which used its R 2 ϩ subunit to complete the double-strand break.…”

Section: Resultsmentioning

confidence: 99%

“…The BbvCI restriction enzyme, and its R 1 ϩ R 2 Ϫ mutant, were purified as described in ref. 30, and their concentrations, in terms of the M r 64053 R 1 R 2 heterodimer, were determined from A 280 readings (31). Other enzymes were from New England Biolabs.…”

Section: Methodsmentioning

confidence: 99%

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Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

Gowers

Wilson

Halford

2005

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

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223

257

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Proteins that act at specific DNA sequences bind DNA randomly and then translocate to the target site. The translocation is often ascribed to the protein sliding along the DNA while maintaining continuous contact with it. Proteins also can move on DNA by multiple cycles of dissociation͞reassociation within the same chain. To distinguish these pathways, a strategy was developed to analyze protein motion between DNA sites. The strategy reveals whether the protein maintains contact with the DNA as it transfers from one site to another by sliding or whether it loses contact by a dissociation͞reassociation step. In reactions at low salt, the test protein stayed on the DNA as it traveled between sites, but only when the sites were <50 bp apart. Transfers of >30 bp at in vivo salt, and over distances of >50 bp at any salt, always included at least one dissociation step. Hence, for this enzyme, 1D sliding operates only over short distances at low salt, and 3D dissociation͞ reassociation is its main mode of translocation.DNA-protein interaction ͉ recognition sequence ͉ restriction enzyme

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

Gowers

Wilson

Halford

2005

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

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223

257

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“…A motor-stator duplex contains the recognition site for a nicking restriction enzyme that catalyses hydrolysis of a specific backbone linkage of the stator 20 . The released energy drives movement of the motor from the cut stator to an adjacent intact stator 6 .…”

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confidence: 99%

Direct observation of stepwise movement of a synthetic molecular transporter

et al. 2011

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Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor 1-12 and fuel [13][14][15] , all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold 16 , and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor. Real-time atomic force microscopy allows direct observation of individual steps of a single motor, revealing mechanistic details of its operation. This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.An effective linear molecular motor must traverse its track without dissociating [1][2][3][4][5][6][7]10,12 and run unidirectionally without external intervention [4][5][6][7][8][9][10][11][12] . Directionality may be imposed by the sequential addition of DNA instructions 1-3 or, for autonomous motors, by modifying the track sites that have been visited 5,6,12 , by coupling motion to a unidirectional reaction cycle 4,9,12 or by coordinating the conformation changes of different parts of the motor 11,12 . DNA motors that satisfy all these criteria have typically been demonstrated on tracks that allow only 1-3 steps, although a stochastic DNA 'spider' with many legs has been shown to move longer distances by biased diffusion 17 along a 100 nm track 18 .We have investigated the motion of a simple directional and processive motor fuelled by DNA hydrolysis 6 along an extended track consisting of a one-dimensional array of single-stranded attachment sites (stators), separated by 6 nm. An extended track of 15 identical stators, flanked with special start and stop stators 6 , was assembled on a rectangular DNA origami tile measuring 100 nm × 70 nm (ref. 16; Fig. 1, Supplementary Figs S1, S2). The tile comprises a 7,249-nucleotide (nt) single-stranded DNA template (genome of bacteriophage M13) hybridized to short synthetic staple strands such that the final tile consists of a raft of 24 parallel double helices tethered by the crossover of staples. Two tile designs were used. The helices of tile type A are crosslinked at 16 bp intervals, creating slight underwinding (10.7 bp per turn), which is expected to lead to a global right-handed twisting of the tile 19 . Tile type B is designed to reduce this twist: the average distance between crossovers is 15.6 bp (giving 10.4 bp per turn). The centre and ends of each staple are positioned on opposite surfaces of the tile. Selected staples were extended to include either the 22-nt stator sequence at the 5 ′ end or a hairpin at the centre 16 (Fig. 1a). Stators hybridized to complementary motor strands are visible in atomic force microscope (AFM) images...

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A Free‐Running DNA Motor Powered by a Nicking Enzyme

Bath¹,

Green²,

Turberfield³

2005

Angewandte Chemie

119

116

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Herein, we present a simple linear motor, built from DNA and a restriction enzyme, which moves a DNA cargo in discrete steps along a DNA track. Movement is powered by a nicking enzyme that cuts the track. Damage to the track in the wake of the cargo imposes directionality.The specificity of base-pairing, the rigidity of short segments of the double helix, and the flexibility of singlestranded segments make DNA an ideal material for construction of nanometer-sized mechanical devices. [1][2][3][4][5][6][7] Such devices can generate forces of the order of picoNewtons, [2] in the same range as the forces developed by single-molecule

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Site-Specific DNA-nicking Mutants of the Heterodimeric Restriction Endonuclease R.BbvCI

Cited by 81 publications

References 39 publications

Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

Direct observation of stepwise movement of a synthetic molecular transporter

A Free‐Running DNA Motor Powered by a Nicking Enzyme

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