Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements.

Hager, Paul W.; Reich, Norbert O.; Day, John; Coche, Th.; Boyer, Herbert W.; Rosenberg, John M.; Greene, Patricia J.

doi:10.1016/s0021-9258(18)45770-5

Cited by 16 publications

(4 citation statements)

References 33 publications

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“…Finally, the lack of catalytic processivity for the MTase means that other canonical sites on the viral genome will remain unprotected and can be recognized and cleaved by the ENase. Our kinetic analysis of the EcoRI MTase, coupled with past work on the ENase (Jack et al, 1982;Terry et al, 1985;Hager et al, 1990), provide a satisfying rationale for how this primordial "immune" system might function. Whether this model is generally applicable awaits the characterization of other restriction-modification systems.…”

Section: Discussionmentioning

confidence: 94%

“…b All values are apparent values determined at 37 °C under the following conditions: 100 mM Tris, pH 8.0, 10 mM EDTA, 200 µg of BSA/mL, and 10 mM dithiothreitol. DNA concentrations varied from 0.5 to 10 times Km at a saturating AdoMet concentration of 1.5 µM and 0.05 nM MTase (Reich & Mashhoon, 1991) Whether an unmodified site is methylated or cut is essentially determined by which enzyme arrives at the site first since catalysis by both enzymes is diffusion controlled: a k cat /K m of 1.3 × 10 7 s -1 M -1 has been reported for the ENase with pBR322 as a substrate (Hager et al, 1990). Since both enzymes recognize the same DNA sequence and modification of this site by the MTase blocks cleavage by the ENase, a single-turnover assay was developed to assess the relative efficiencies of site location and catalysis by the two enzymes through direct competition.…”

Section: Mtase Does Not Methylate Multiple Sites On the Same Dna Mole...mentioning

confidence: 99%

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Contribution of Facilitated Diffusion and Processive Catalysis to Enzyme Efficiency: Implications for the EcoRI Restriction−Modification System

Surby

Reich

1996

Biochemistry

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The contribution of nonspecific DNA to enzyme efficiency (k(cat)/K(m)) is described for a sequence-specific DNA-modifying enzyme. Our investigation focuses on the EcoRI DNA methyltransferase which transfers a methyl group from the cofactor S-adenosylmethionine to the second adenine in the double-stranded DNA sequence GAATTC. k(cat)/K(m) increases 4-fold as DNA length increases from 14 to 429 base pairs and increases 2-fold as the distance from the site to the nearest end is increased from 29 to 378 base pairs. No changes in k(cat)/K(m) result from further increases in either case. A facilitated diffusion mechanism is proposed in which the methyltransferase scans an average of <400 base pairs prior to dissociation from a DNA molecule. The methyltransferase was found to methylate two sites on a single DNA molecule in a distributive rather than a processive manner, suggesting that the enzyme dissociates from the DNA prior to release of the reaction product S-adenosylhomocysteine. A direct competition experiment with the EcoRI endonuclease shows the methyltransferase to be slightly more efficient at specific site location and catalysis. A rationale for the role of facilitated diffusion in this type II restriction-modification system is proposed.

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Section: Discussionmentioning

confidence: 94%

Section: Mtase Does Not Methylate Multiple Sites On the Same Dna Mole...mentioning

confidence: 99%

Contribution of Facilitated Diffusion and Processive Catalysis to Enzyme Efficiency: Implications for the EcoRI Restriction−Modification System

Surby

Reich

1996

Biochemistry

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“…If each hydrogen bond contributed -1.5 kcal/mol, the loss of 4 bonds should reduce specific binding by a factor of 30 000. In the EcoRI enzyme, mutations at residues that contact the DNA bases do indeed reduce the specific binding constant by factors of this sort (Alves et al, 1989;Hager et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

“…Thielking et al (1991) reported that partially purified preparations of N185A had no DNA cleavage activity in the presence of MgCl2, in contrast to the very low activity observed here with the purified enzyme. The discrepancy may be due to the fact that Thielking et al (1991) used a linear substrate (phage X DNA), rather than supercoiled DNA, so they would have not detected products until both strands of the DNA had been cut at the same site; even with high concentrations of N185A, only a fraction of the DNA will have double-strand breaks after 24 h. A number of mutants of EcoRI also cleave DNA by successive single-strand breaks, under conditions where wt EcoRI makes double-strand breaks (Alves et al, 1989;Needels et al, 1989;Heitman & Model, 1990a;Hager et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

EcoRV restriction endonuclease: communication between DNA recognition and catalysis

Vipond²,

Halford³

1992

Biochemistry

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A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.

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How the EcoRI endonuclease recognizes and cleaves DNA

Heitman

1992

BioEssays

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One popular recombinant DNA tool is the EcoRI endonuclease, which cleaves DNA at GAATTC sites and serves as a paradigm for sequence specific DNA-enzyme interactions. The recently revised X-ray crystal structure of an EcoRI-DNA complex reveals EcoRI employs novel DNA recognition motifs, a four alpha-helix bundle and two extended chains, which project into the major groove to contact substrate purines and pyrimidines. Interestingly, pyrimidine contacts had been predicted based on genetic and biochemical studies. Current work focuses on the EcoRI active site structure, enzyme and substrate conformational changes during catalysis, and host-restriction system interactions.

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Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements.

Cited by 16 publications

References 33 publications

Contribution of Facilitated Diffusion and Processive Catalysis to Enzyme Efficiency: Implications for the EcoRI Restriction−Modification System

Contribution of Facilitated Diffusion and Processive Catalysis to Enzyme Efficiency: Implications for the EcoRI Restriction−Modification System

EcoRV restriction endonuclease: communication between DNA recognition and catalysis

How the EcoRI endonuclease recognizes and cleaves DNA

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