The Kinetic Mechanism of EcoRI Endonuclease
نویسندگان
چکیده
منابع مشابه
The kinetic mechanism of EcoRI endonuclease.
Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with K(m) and k(cat) increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23 M. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has a...
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The mechanism of EcoRI endonuclease is substrate dependent. At 37 degrees dissociation of the enzyme-Form II DNA intermediates of ColE1 DNA and bacteriophage G4 RFI DNA is negligible. Therefore, both DNA strands with in the EcoRI sequence are cleaved during a single binding event. However, double strand cleavage of SV40 DNA occurs without dissociation of the enzyme in only 75% of the catalytic ...
متن کاملSpecificity of substrate recognition by the EcoRI restriction endonuclease.
The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence d(N-G-A-A-T-T-C-N)/d(N-C-T-T-A-A-G-N) when the ionic strength is high, 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 ...
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Studies presented here demonstrate that heparin inhibits EcoRI endonuclease cleavage of DNA whereas related proteoglycans show no effect. The inhibition occurs at particular EcoRI sites that are near or overlap with palindromic sequences in the murine lambda 5 and Lyt-2 genes. Endogenous heparin from peritoneal mast cells co-isolates with DNA and inhibits digestion of peritoneal cell DNA at the...
متن کاملArtificial mobile DNA element constructed from the EcoRI endonuclease gene.
There exist several examples of mobile group I introns. These introns appear to use a straightforward mechanism to achieve highly site-specific and efficient insertion into homologous intronless genes. Because the only intron-specific function required by the prevailing model for the mechanism of intron mobility is the introduction of a site-specific double-stranded break in the intronless reci...
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ژورنال
عنوان ژورنال: Journal of Biological Chemistry
سال: 1999
ISSN: 0021-9258
DOI: 10.1074/jbc.274.45.31896