Linked Life Data

10079077

Source:http://linkedlifedata.com/resource/pubmed/id/10079077

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
1999-4-15
pubmed:abstractText
Oligonucleotides that contain a single modified pyrimidine, i.e., thymine glycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were synthesized in order to investigate the substrate specificity and the excision mechanism of two Escherichia coli repair enzymes: endonuclease III and formamidopyrimidine DNA glycosylase (Fpg). Three techniques of analysis were employed. A gas chromatography-mass spectrometry (GC-MS) assay with HPLC prepurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage. Values of Vm/Km constants lead to the conclusion that the substrates are processed by endonuclease III with the following preference: Tg >> 5-OHC > DHT. This confirms that Tg is an excellent substrate for endonuclease III. Fpg-mediated cleavage of the 5-OHC-containing oligonucleotide is processed at the same rate than endonuclease III. Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificity of this enzyme to a new modified pyrimidine. While 5-OHC-containing oligonucleotides are cleaved by the two enzymes, no or a small amount of the modified base was found to be released, as determined by GC-MS. From these data it may be suggested that 5-OHC could be modified during its enzymatic excision. Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-OHC- and DHT-containing oligonucleotides showed that endonuclease III cleaves the DNA backbone mainly through a hydrolytic process and that no beta-elimination product was detected.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/5,6-dihydrothymine, http://linkedlifedata.com/resource/pubmed/chemical/5-hydroxycytosine, http://linkedlifedata.com/resource/pubmed/chemical/Cytosine, http://linkedlifedata.com/resource/pubmed/chemical/DNA-Formamidopyrimidine Glycosylase, http://linkedlifedata.com/resource/pubmed/chemical/DNA-formamidopyrimidine..., http://linkedlifedata.com/resource/pubmed/chemical/Deoxyribonuclease (Pyrimidine Dimer), http://linkedlifedata.com/resource/pubmed/chemical/Endodeoxyribonucleases, http://linkedlifedata.com/resource/pubmed/chemical/Escherichia coli Proteins, http://linkedlifedata.com/resource/pubmed/chemical/N-Glycosyl Hydrolases, http://linkedlifedata.com/resource/pubmed/chemical/NTH protein, E coli, http://linkedlifedata.com/resource/pubmed/chemical/Oligodeoxyribonucleotides, http://linkedlifedata.com/resource/pubmed/chemical/Thymine, http://linkedlifedata.com/resource/pubmed/chemical/thymine glycol
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
16
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3335-44
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects.
pubmed:affiliation
Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, Département de Recherche Fondamentale sur la Matière Condensée, CEA Grenoble, France.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't