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A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

Emily C. Sheppard, Sally Rogers, View ORCID ProfileNicholas J. Harmer, View ORCID ProfileRichard Chahwan
doi: https://doi.org/10.1101/548628
Emily C. Sheppard
1Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
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Sally Rogers
1Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
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Nicholas J. Harmer
1Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
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Richard Chahwan
1Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
2Institute of Experimental Immunology, University of Zurich, 8057 Zurich, CH
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  • ORCID record for Richard Chahwan
  • For correspondence: richard.chahwan@uzh.ch
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Abstract

DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted February 14, 2019.
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A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity
Emily C. Sheppard, Sally Rogers, Nicholas J. Harmer, Richard Chahwan
bioRxiv 548628; doi: https://doi.org/10.1101/548628
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A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity
Emily C. Sheppard, Sally Rogers, Nicholas J. Harmer, Richard Chahwan
bioRxiv 548628; doi: https://doi.org/10.1101/548628

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