A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity

Maryati, Marayti and Kaur, Ishwinder and Jadhav, Gopal P. and Olotu-Umoren, Loyin and Oveh, Blessing and Hashmi, Lubna and Fischer, Peter M. and Winkler, G. Sebastiaan (2013) A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. Nucleic Acids Research . pp. 1-10. ISSN 0305-1048

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Abstract

In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in posttranscriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed

to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease–exonuclease–phosphatase domain. Both domains require the presence of two Mg2+ ions in the active site. To facilitate the biochemical analysis of deadenylase enzymes, we have

developed a fluorescence-based deadenylase assay. The assay is based on end-point measurement, suitable for quantitative analysis and can be adapted for 96- and 384-well microplate formats. We demonstrate the utility of the assay by screening a chemical compound library, resulting

in the identification of non-nucleoside inhibitors of the Caf1/CNOT7 enzyme, a catalytic subunit of the Ccr4–Not deadenylase complex. These compounds may be useful tools for the biochemical analysis of the Caf1/CNOT7 deadenylase

subunit of the Ccr4–Not complex and indicate the feasibility of developing selective inhibitors of deadenylase enzymes using the fluorescencebased assay.

Item Type: Article
Additional Information: Online version
Schools/Departments: University of Nottingham UK Campus > Faculty of Science > School of Pharmacy
Identification Number: https://doi.org/10.1093/nar/gkt972
Depositing User: de Sousa, Mrs Shona
Date Deposited: 16 Apr 2014 10:33
Last Modified: 15 Sep 2016 02:28
URI: http://eprints.nottingham.ac.uk/id/eprint/2323

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