Rapid Reaction Analysis of the Catalytic Cycle of the EcoRV Restriction Endonuclease

Baldwin, Geoff; Vipond, IB; Halford, SE

doi:10.1021/bi00002a038

Cited by 82 publications

(130 citation statements)

References 18 publications

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“…This second order assembly is likely to be the slowest step in the pathway and the reason why DNA containing only a single GAAGA site is poorly cut. The reaction cannot, as is possible with orthodox type II restriction endonucleases (41)(42)(43)(44)(45)(46)(47), be fitted to a single exponential to give a rate constant with units of time Ϫ1 . However, the simple comparison of the half-life seen with one-and twosite plasmids clearly indicates the strong preference of MboII for the latter.…”

Section: Discussionmentioning

confidence: 99%

DNA Binding and Recognition by the IIs Restriction Endonuclease MboII

Soundararajan¹,

Chang²,

Morgan³

et al. 2002

Journal of Biological Chemistry

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The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca 2؉ is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca 2؉ , possibly a (MboII) 2 -(DNA) 2 complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme) 2 -(DNA) 2 complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.The best characterized type II restriction endonucleases, exemplified by EcoRI, EcoRV, BamHI, and PvuII, are homodimers that cut DNA within palindromic target sites, from 4 to 8 base pairs in size (1-3). Generally one protein subunit recognizes one-half of the palindromic sequence and, in an identical arrangement, the second subunit interacts with the other half, resulting in a symmetric protein-DNA complex. Each of the protein subunits contains a catalytic site, enabling both strands of the DNA to be cut, often in a highly concerted reaction. However, many type II restriction endonucleases do not correspond to these simple paradigms. One category, classified as type IIs systems (4), recognizes nonpalindromic DNA sequences, between 4 and 7 base pairs in length and cut up to 20 bases outside their target sites. A nonsymmetrical DNA target site does not allow for simple recognition using a protein with a homodimeric subunit arrangement, and indeed, most type IIs restriction enzymes appear to be monomeric (5-7). As a monomeric enzyme will contain only a single active site, there is no straightforward way to achieve the hydrolysis of both DNA strands. FokI, the best studied type IIs enzyme, recognizes GGATG [9/13] sites (the numbers in brackets refer to the distance to the cutting site on the top and bottom strands, respectively) and contains separate DNA recognition and cutting domains (8). A crystal structure shows FokI bound to DNA as a monomer with the recognition domain interacting with the GGATG bases; the cutting domain is tightly associated with the recognition domain and too far from the scissile phosphates to cause hydrolysis (9). A second structure, of the free enzyme, shows a dimer in which the dimerization interface is com...

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Section: Discussionmentioning

confidence: 99%

DNA Binding and Recognition by the IIs Restriction Endonuclease MboII

Soundararajan¹,

Chang²,

Morgan³

et al. 2002

Journal of Biological Chemistry

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“…The similarity of the two rate constants is in agreement with the assertion that the trajectories comprise molecular events like DNA bending, DNA hydrolysis, and product release, but not DNA binding, which is expected to be much slower in case of the 30-bp spacer because of steric hindrance. The catalytic cycle of EcoRV with short (7-14 bp) substrate DNAs has been investigated in bulk by using FRET, stopped-flow fluorescence, fluorescence anisotropy, and quench-flow methods (46,48,49). These studies found that the turnover rate k cat is limited by rates of DNA hydrolysis and product release (Fig.…”

Section: Dynamics Of Ecorv-catalyzed Dna Cleavage As Measured With Plmentioning

confidence: 99%

Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes

Reinhard

Sheikholeslami

Mastroianni

et al. 2007

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

270

328

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Pairs of Au nanoparticles have recently been proposed as ''plasmon rulers'' based on the dependence of their light scattering on the interparticle distance. Preliminary work has suggested that plasmon rulers can be used to measure and monitor dynamic distance changes over the 1-to 100-nm length scale in biology. Here, we substantiate that plasmon rulers can be used to measure dynamical biophysical processes by applying the ruler to a system that has been investigated extensively by using ensemble kinetic measurements: the cleavage of DNA by the restriction enzyme EcoRV. Temporal resolutions of up to 240 Hz were obtained, and the end-to-end extension of up to 1,000 individual dsDNA enzyme substrates could be simultaneously monitored for hours. The kinetic parameters extracted from our singlemolecule cleavage trajectories agree well with values obtained in bulk through other methods and confirm well known features of the cleavage process, such as DNA bending before cleavage. Previously unreported dynamical information is revealed as well, for instance, the degree of softening of the DNA just before cleavage. The unlimited lifetime, high temporal resolution, and high signal/noise ratio make the plasmon ruler a unique tool for studying macromolecular assemblies and conformational changes at the single-molecule level.

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“…Biophysical experiments Endonuclease activity was assayed in 50 mM triethanolamine buffer pH 7.5, 10 mM Mg 2+ at 25°C with 5 µM protein and 2 µM DNA by following the hyperchromic shift resulting from the cleavage of a double-stranded oligonucleotide (after Baldwin et al 46 ). A 12 mer dsDNA palindromic sequence 5'-GACGATATCGTC-3' (reannealed following prior heating to 80°C) was used as substrate and its hydrolysis monitored by time dependent increase of A 260 after mixing the protein with DNA using a π* stoppedflow CD spectrophotometer in the absorbance mode (Applied Photophysics).…”

Section: Planar Lipid Bilayers and Single-channel Recordings Bilayersmentioning

confidence: 99%

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

et al. 2002

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Article:Mosbahi, Khédidja, Lemaître, Christelle, Mobasheri, Hamid et al. (6 more authors) (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology. pp. 476-484. ISSN 1545-9985 https://doi.org/10.1038/nsb797 eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. White Rose Consortium ePrints Repositoryhttp://eprints.whiterose.ac.uk/ This is an author produced version of a paper published in Nature Structural Biology. This paper has been peer-reviewed but does not include final publisher proof-corrections or journal pagination.White Rose Repository URL for this paper: http://eprints.whiterose.ac.uk/archive/00001066/ Citation for the published paper Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology, 9 (6). pp. 476-484. Citation for this paperTo refer to the repository paper, the following format may be used: Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) Colicin E9 is a 60 kDa toxin that is normally released from colicinogenic bacteria in the form of a heterodimeric complex with its 9.5 kDa immunity protein, Im9 (ref. 14). The immunity protein protects the colicin-producing bacterium from the activity of its own toxin but is jettisoned on entry of the colicin into a susceptible cell 15 . Hence, the form of the toxin tested in the bilayer experiments had the immunity protein removed (see Materials andMethods section). Previous work from our laboratory has shown that this form of the toxin retains complete biological activity 14 . Immunity-free colicin E9 (2 nM) was added to the cis chamber of a bilayer apparatus in 10 mM Tris/HCl buffer at pH 7.5, containing 0.1 M NaCl and 10 mM CaCl 2 , and a potential difference (p.d.) applied across the membrane. Random, fluctuating current was observed that showed evidence of opening and closing events with conductance of the order of ~100 pS, although larger conductance states were also seen ( Fig. 1a). In order to identify the region(s) of the protein responsible for this a...

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Rapid Reaction Analysis of the Catalytic Cycle of the EcoRV Restriction Endonuclease

Cited by 82 publications

References 18 publications

DNA Binding and Recognition by the IIs Restriction Endonuclease MboII

DNA Binding and Recognition by the IIs Restriction Endonuclease MboII

Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

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