Specific DNA Recognition by EcoRV Restriction Endonuclease Induced by Calcium Ions

Vipond, IB; Halford, SE

doi:10.1021/bi00004a002

Cited by 131 publications

(145 citation statements)

References 25 publications

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“…The decline in processivity thus can be assigned to the effect of salt on weakening nonspecific binding. The affinities and the salt dependency of nonspecific DNA binding by BbvCI are comparable with other restriction enzymes (34)(35)(36)(37).…”

Section: Resultsmentioning

confidence: 54%

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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: 54%

“…The key factor in determining the mean path length for 1D diffusion by a protein on DNA is its affinity for nonspecific DNA (8)(9)(10)(11). In this respect, BbvCI is comparable with other restriction enzymes (34)(35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

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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“…However, Ca 2ϩ , which does not promote DNA hydrolysis, has been extensively used as a Mg 2ϩ surrogate. With orthodox type II restriction endonucleases, Ca 2ϩ is commonly observed to increase the affinity for cognate DNA sequences often by considerable factors of up to 10 4 (35)(36)(37)(38)(39)(40). Recently it has been shown that the production of dimeric FokI complexes requires the presence of a divalent metal ion.…”

Section: Discussionmentioning

confidence: 99%

“…with R.EcoRV (35,36), R.MunI (37), R.PvuII (38), R.Cfr10I (39), and R.BamHI (40). Recently, Ca 2ϩ has been found to be essential for the formation of (FokI) 2 -(DNA) 2 complexes (12).…”

Section: Fig 2 Interaction Of the Mboii Restriction Endonuclease Wimentioning

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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“…Similar to EcoRV, a stable complex was formed in the presence of 10 mM Ca 2ϩ , although the activity of PvuII using Ca 2ϩ as a cofactor was negligible ( Fig. 2 and data not shown) (41). Several attempts to visualize specific PvuII-DNA complexes in the absence of divalent ions were attempted unsuccessfully (data not shown).…”

Section: Analysis Of Interaction Between Pvuii and Its Specific Bindimentioning

confidence: 93%

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

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The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5 -CAGCTG-3 ) through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA-or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.The structures of the five type II endonucleases determined by x-ray diffraction, EcoRI (1), BamHI (2, 3), EcoRV (4, 5), PvuII (6, 7) and Cfr10I (8), share substantial elements of similarity. Analysis of the enzymes complexed to DNA, where known, suggests a preliminary classification into two different groups (9). The endonucleases that produce 5Ј-overhanging ends, EcoRI and BamHI, approach the DNA from the major groove, where most of their base-specific contacts are made. The endonucleases that produce blunt-ended DNA products, EcoRV and PvuII, approach the DNA from the minor groove side and establish contacts in the major groove by wrapping around the DNA. Structure-function studies have been limited to a small number of type II enzymes. Years of studies have produced an extensive amount of information about EcoRI (10 -18), EcoRV (18 -24), and NaeI (25) and, more recently, BamHI (26,27). These studies have focused on the identification and analysis of residues involved in catalysis, testing alternative mechanisms of catalysis, and understanding how a specific DNA sequence is recognized. Attempts to alter the sequence specificity have proven to be difficult, with successful examples limited to mutants showing relaxed specificity (10,11,28) or displaying activity toward DNA substituted with unnatural bases (15,29). One explanation for this difficulty is that a change in specificity not only requires recognition of a different DNA sequence, but also requires that the linkage between recognition and catalysis be retained. A class of mutations that might illuminate this linkage is the catalytic mutations, where specific DNA binding is retained but catalysis is reduced (26, 30). In particular, mutants in this class that are outside the catalytic center might be deficient in the linkage between recognition and ca...

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Specific DNA Recognition by EcoRV Restriction Endonuclease Induced by Calcium Ions

Cited by 131 publications

References 25 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

DNA Binding and Recognition by the IIs Restriction Endonuclease MboII

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

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