2.50
Hdl Handle:
http://hdl.handle.net/10541/78137
Title:
A molecular beacon assay for measuring base excision repair activities.
Authors:
Maksimenko, Andrei; Ishchenko, Alexander A; Sanz, Guenhaël; Laval, Jacques; Elder, Rhoderick H; Saparbaev, Murat K
Abstract:
The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.
Affiliation:
BioAlliance Pharma SA, 59, Bvd du Général Martial Valin, 75015 Paris, France.
Citation:
A molecular beacon assay for measuring base excision repair activities. 2004, 319 (1):240-6 Biochem. Biophys. Res. Commun.
Journal:
Biochemical and Biophysical Research Communications
Issue Date:
18-Jun-2004
URI:
http://hdl.handle.net/10541/78137
DOI:
10.1016/j.bbrc.2004.04.179
PubMed ID:
15158468
Type:
Article
Language:
en
ISSN:
0006-291X
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorMaksimenko, Andrei-
dc.contributor.authorIshchenko, Alexander A-
dc.contributor.authorSanz, Guenhaël-
dc.contributor.authorLaval, Jacques-
dc.contributor.authorElder, Rhoderick H-
dc.contributor.authorSaparbaev, Murat K-
dc.date.accessioned2009-08-21T10:32:03Z-
dc.date.available2009-08-21T10:32:03Z-
dc.date.issued2004-06-18-
dc.identifier.citationA molecular beacon assay for measuring base excision repair activities. 2004, 319 (1):240-6 Biochem. Biophys. Res. Commun.en
dc.identifier.issn0006-291X-
dc.identifier.pmid15158468-
dc.identifier.doi10.1016/j.bbrc.2004.04.179-
dc.identifier.urihttp://hdl.handle.net/10541/78137-
dc.description.abstractThe base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.en
dc.language.isoenen
dc.subject.meshAnimals-
dc.subject.meshCell-Free System-
dc.subject.meshDNA-
dc.subject.meshDNA Damage-
dc.subject.meshDNA Glycosylases-
dc.subject.meshDNA Repair-
dc.subject.meshDNA-(Apurinic or Apyrimidinic Site) Lyase-
dc.subject.meshGenetic Techniques-
dc.subject.meshHela Cells-
dc.subject.meshHumans-
dc.subject.meshMice-
dc.subject.meshNIH 3T3 Cells-
dc.subject.meshNucleic Acid Conformation-
dc.subject.meshOligonucleotides-
dc.subject.meshOxygen-
dc.subject.meshSaccharomyces cerevisiae-
dc.subject.meshSpectrometry, Fluorescence-
dc.subject.meshTime Factors-
dc.titleA molecular beacon assay for measuring base excision repair activities.en
dc.typeArticleen
dc.contributor.departmentBioAlliance Pharma SA, 59, Bvd du Général Martial Valin, 75015 Paris, France.en
dc.identifier.journalBiochemical and Biophysical Research Communicationsen

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