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Quantitative Analysis of WRN Exonuclease Activity by Isotope Dilution Mass Spectrometry

Quantitative Analysis of WRN Exonuclease Activity by Isotope Dilution Mass Spectrometry

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MANGERICH, Aswin, Sebastian VEITH, Oliver POPP, Rita MARTELLO, Vilhelm BOHR, Alexander BÜRKLE, Jörg FAHRER, 2012. Quantitative Analysis of WRN Exonuclease Activity by Isotope Dilution Mass Spectrometry. In: Mechanisms of Ageing and Development. 133(8), pp. 575-579. ISSN 0047-6374. eISSN 1872-6216

@article{Mangerich2012-08Quant-19706, title={Quantitative Analysis of WRN Exonuclease Activity by Isotope Dilution Mass Spectrometry}, year={2012}, doi={10.1016/j.mad.2012.06.005}, number={8}, volume={133}, issn={0047-6374}, journal={Mechanisms of Ageing and Development}, pages={575--579}, author={Mangerich, Aswin and Veith, Sebastian and Popp, Oliver and Martello, Rita and Bohr, Vilhelm and Bürkle, Alexander and Fahrer, Jörg} }

<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/19706"> <dc:creator>Fahrer, Jörg</dc:creator> <dcterms:issued>2012-08</dcterms:issued> <dc:creator>Martello, Rita</dc:creator> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2012-08-23T13:06:07Z</dc:date> <dc:contributor>Veith, Sebastian</dc:contributor> <dc:contributor>Bürkle, Alexander</dc:contributor> <dc:contributor>Fahrer, Jörg</dc:contributor> <dcterms:bibliographicCitation>Publ. in: Mechanisms of Ageing and Development ; 133 (2012), 8. - S. 575-579</dcterms:bibliographicCitation> <dc:rights>deposit-license</dc:rights> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2012-08-23T13:06:07Z</dcterms:available> <dcterms:abstract>Werner syndrome is a disorder characterized by a premature aging phenotype. The disease is caused by mutations in the WRN gene which encodes a DNA helicase/exonuclease which is involved in multiple aspects of DNA metabolism. Current methods mostly rely on radiometric techniques to assess WRN exonuclease activity. Here we present an alternative, quantitative approach based on non-radioactive isotope dilution mass spectrometry (LC-MS/MS). A oligoduplex substrate mimicking the telomeric sequence was used for method development. Released nucleotides, which correlate with the degree of oligoduplex degradation, were dephosphorylated, purified, and quantified by LC-MS/MS. Heavy-isotope-labeled internal standards were used to account for technical variability. The method was validated in terms of reproducibility, time-course and concentration-dependency of the reaction. As shown in this study, the LC-MS/MS method can assess exonuclease activity of WRN mutants, WRN's substrate and strand specificity, and modulatory effects of WRN interaction partners and posttranslational modifications. Moreover, it can be used to analyze the selectivity and processivity of WRN exonuclease and allows the screening of small molecules for WRN exonuclease inhibitors. Importantly, this approach can easily be adapted to study nucleases other than WRN. This is of general interest, because exonucleases are key players in DNA metabolism and aging mechanisms.</dcterms:abstract> <dc:creator>Veith, Sebastian</dc:creator> <dc:language>eng</dc:language> <dc:contributor>Martello, Rita</dc:contributor> <dc:creator>Mangerich, Aswin</dc:creator> <dcterms:title>Quantitative Analysis of WRN Exonuclease Activity by Isotope Dilution Mass Spectrometry</dcterms:title> <dc:creator>Popp, Oliver</dc:creator> <dcterms:rights rdf:resource="http://nbn-resolving.org/urn:nbn:de:bsz:352-20140905103605204-4002607-1"/> <dc:contributor>Popp, Oliver</dc:contributor> <dc:contributor>Mangerich, Aswin</dc:contributor> <dc:contributor>Bohr, Vilhelm</dc:contributor> <dc:creator>Bürkle, Alexander</dc:creator> <dc:creator>Bohr, Vilhelm</dc:creator> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/19706"/> </rdf:Description> </rdf:RDF>

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