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

Maryati, Marayti; Kaur, Ishwinder; Jadhav, Gopal P.; Olotu-Umoren, Loyin; Oveh, Blessing; Hashmi, Lubna; Fischer, Peter M.; Winkler, G. Sebastiaan

Authors

Marayti Maryati

Ishwinder Kaur

Gopal P. Jadhav

Loyin Olotu-Umoren

Blessing Oveh

Lubna Hashmi

Peter M. Fischer

G. Sebastiaan Winkler



Abstract

In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in post-transcriptional 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 fluorescence-based assay. © The Author(s) 2013.

Journal Article Type Article
Publication Date Mar 1, 2014
Journal Nucleic Acids Research
Print ISSN 0305-1048
Electronic ISSN 1362-4962
Publisher Oxford University Press
Peer Reviewed Peer Reviewed
Volume 42
Issue 5
APA6 Citation Maryati, M., Kaur, I., Jadhav, G. P., Olotu-Umoren, L., Oveh, B., Hashmi, L., …Winkler, G. S. (2014). A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. Nucleic Acids Research, 42(5), https://doi.org/10.1093/nar/gkt972
DOI https://doi.org/10.1093/nar/gkt972
Publisher URL https://academic.oup.com/nar/article/42/5/e30/1063553
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0
Additional Information Online version

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