Linked Life Data

7662656

Source:http://linkedlifedata.com/resource/pubmed/id/7662656

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
34
pubmed:dateCreated
1995-10-6
pubmed:abstractText
The DNA-binding properties of the EcoRV restriction endonuclease and modification methyltransferase with their recognition sequence (GATATC) were analyzed using the electrophoretic band-shift assay. It has previously been observed that the endonuclease does not bind specifically to GATATC sequences in the absence of the essential cofactor Mg2+. To investigate any possible roles for Mg2+ in promoting specific DNA binding, a set of hydrolysis-resistant oligonucleotide substrates were synthesized that contained either phosphate (phosphorothioate, 3'-S-phosphorothiolate), sugar (4'-thiothymidine), or base (7-deaza-2'-deoxyadenosine) modifications. However, it was found that none of these were specifically bound by the endonuclease in either the absence or the presence of Mg2+. In contrast, the methylase bound to GATATC sequences much more strongly than to nonspecific sites, and it was possible to observe the formation of enzyme--DNA complexes by gel retardation. Binding to GATATC sequences was increased by the addition of sinefungin, a nonreactive analogue of the essential cofactor S-adenosyl-L-methionine (AdoMet). Presumably this also occurs with AdoMet although methylation and turnover prevented its direct observation. In the presence of sinefungin the strongest binding was observed with hemimethylated EcoRV sequences (Kd = 11-13 nM), and unmethylated DNA was bound less well (Kd = 46 nM). Specific, albeit weaker binding was also seen with the dimethylated product (Kd = 143 nM). A difference in electrophoretic mobility was observed between enzyme-substrate and enzyme-product complexes suggestive of structural differences between them. The Kapp value found for sinefungin, with the hemimethylated EcoRV sequence, was 10.9 mM.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
29
pubmed:volume
34
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
10724-33
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Sequence-specific binding of DNA by the EcoRV restriction and modification enzymes with nucleic acid and cofactor analogues.
pubmed:affiliation
Department of Biochemistry, University of Southampton, U.K.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't