Linked Life Data

7669777

Source:http://linkedlifedata.com/resource/pubmed/id/7669777

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
35
pubmed:dateCreated
1995-10-13
pubmed:abstractText
In order to investigate the accuracy of the EcoRV restriction endonuclease, we have synthesized a set of double-stranded oligodeoxynucleotides comprising the canonical recognition sequence, the 9 star sequences (i.e., sequences deviating by one base pair from the canonical sequence), and the 18 mismatch sequences (i.e. sequences deviating by one base from the canonical sequence). For each individual single strand of all these 28 substrates we have measured the rate of phosphodiester bond cleavage under normal buffer conditions. Double-strand cleavage of star substrates is at least 5 orders of magnitude slower than cleavage of the canonical substrate. In contrast, most of the mismatch substrates are accepted more readily. In the absence of the essential cofactor Mg2+, EcoRV binds weakly but equally to the canonical and degenerate substrates, (i.e., KDiss is in the micromolar range). However, the inactive catalytic site mutant D90A in the presence of Mg2+ binds the canonical substrate 1-2 orders of magnitude better than degenerate substrates. Therefore, the EcoRV endonuclease needs the essential cofactor Mg2+ to create thermodynamic discrimination between degenerate and canonical sites. But the main discrimination is kinetically controlled and takes place during cleavage. While in the canonical substrate both single strands are cleaved with an equal velocity, in all other substrates one single strand is cleaved faster than the other one, resulting in a dissociation of the enzyme from the DNA between the two cuts. In vivo this may lead to a repair of the erroneous cleavage site by DNA ligases.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
34
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
11191-7
pubmed:dateRevised
2008-8-29
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Accuracy of the EcoRV restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences.
pubmed:affiliation
Zentrum Biochemie, Medizinische Hochschule Hannover, Germany.
pubmed:publicationType
Journal Article, In Vitro, Research Support, Non-U.S. Gov't