کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1919240 1535625 2012 5 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Quantitative analysis of WRN exonuclease activity by isotope dilution mass spectrometry
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی سالمندی
پیش نمایش صفحه اول مقاله
Quantitative analysis of WRN exonuclease activity by isotope dilution mass spectrometry
چکیده انگلیسی

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.


► Novel bioanalytical method to assess the function of WRN exonuclease.
► Quantitative method based on isotope-dilution mass-spectrometry.
► Validation in terms of reproducibility, kinetics, and concentration-dependency.
► Instrumental to study modulatory effects on WRN exonuclease activity.
► Useful to study specificities and processivity of exonucleases in general.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Mechanisms of Ageing and Development - Volume 133, Issue 8, August 2012, Pages 575–579
نویسندگان
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