EcoRV restriction endonuclease: communication between DNA recognition and catalysis

Cl, Vermote; Vipond, IB; Halford, SE

doi:10.1021/bi00141a019

Cited by 52 publications

(49 citation statements)

References 39 publications

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“…However, the exonuclease activity of the D157N mutant is increased when magnesium chloride is replaced by manganese chloride. A similar increase in activity when Mn 2ϩ replaced Mg 2ϩ has been reported for the active site mutants of EcoRV (18) and BamHI (19) restriction endonucleases. For both the D200N and D157N mutant proteins, the ratio of trinucleotide to dinucleotide products released is lower than that of the wild type protein in Mg 2ϩ ( Fig.…”

Section: Discussionsupporting

confidence: 77%

Identification of Residues of T4 RNase H Required for Catalysis and DNA Binding

Bhagwat

Meara²,

Nossal

1997

Journal of Biological Chemistry

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Bacteriophage T4 encodes a 5Ј-to 3Ј-nuclease with T4 RNase H activity which removes the pentamer RNA primers synthesized during lagging strand DNA replication (1). This T4-encoded nuclease is sufficient for phage DNA synthesis because wild type T4 does not require host enzymes for processing of RNA primers (2). Escherichia coli polymerase I and RNase HI can substitute to some extent if T4 RNase H is deleted, but replication is slower and less accurate in the absence of the T4 enzyme (2).The 5Ј-to 3Ј-nuclease of T4 RNase H degrades both RNA⅐DNA and DNA⅐DNA duplexes, releasing short oligonucleotide products from the 5Ј-end. In the accompanying paper (3) we have shown that T4 RNase H continues to degrade double-stranded DNA until it reaches 8 -11 nucleotides from the 3Ј-end. Although T4 RNase H by itself is a nonprocessive exonuclease, the rate and processivity of the nuclease reaction are increased by the T4 gene 32 single-stranded DNA-binding protein. At the T4 DNA replication fork, single-stranded DNA is covered with the gene 32 protein; hence, T4 RNase H must be acting as a processive exonuclease, removing the RNA primer and some adjacent DNA. Our recent results indicate that this nuclease performs only one round of processive degradation during each lagging strand cycle, removing 10 -50 nucleotides before polymerase elongates the next Okazaki fragment creating a nick sealed by ligase.

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Section: Discussionsupporting

confidence: 77%

Identification of Residues of T4 RNase H Required for Catalysis and DNA Binding

Bhagwat

Meara²,

Nossal

1997

Journal of Biological Chemistry

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“…Rather, all the protein-DNA contacts are intimately connected to each other and additionally to the metal ion binding site and to important catalytic residues. Previously it has been shown that DNA recognition is closely coupled to both metal ion binding (42) and catalysis (43), and this study demonstrates that phosphate groups are also critically involved in these processes.…”

Section: Role Of Phosphates In the Ecorv Endonuclease-catalyzedsupporting

confidence: 58%

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. protein and the GATATC bases, have been probed using alternative sequences (40,42,49), base analogues (13-15, 50 -53), and site-directed mutagenesis (43,54), and many have been shown to be essential for efficient catalysis. The endonuclease also makes extensive contacts to the phosphate backbone (38,39).…”

mentioning

confidence: 99%

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

Thorogood

Grasby

Connolly

1996

Journal of Biological Chemistry

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“…A recent report indicates that EcoRV can bind to DNA in the absence of Ca 2ϩ or Mg 2ϩ under different conditions (44); attempts to visualize a gel shift for PvuII in the absence of metal ions or in the presence of Mg 2ϩ for the catalytically inactive mutants have thus far been unsuccessful. Similar mutagenesis studies have been done on EcoRI (10 -13, 15, 45), EcoRV (18,20,23,24,28), and BamHI (26,27,30). These studies have anticipated or confirmed the predicted roles of residues identified as central from the crystal structures of the respective enzymes.…”

Section: Discussionmentioning

confidence: 63%

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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EcoRV restriction endonuclease: communication between DNA recognition and catalysis

Cited by 52 publications

References 39 publications

Identification of Residues of T4 RNase H Required for Catalysis and DNA Binding

Identification of Residues of T4 RNase H Required for Catalysis and DNA Binding

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

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