PT  - JOURNAL ARTICLE
AU  - Emily C. Sheppard
AU  - Sally Rogers
AU  - Nicholas J. Harmer
AU  - Richard Chahwan
TI  - A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity
AID  - 10.1101/548628
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 548628
4099  - http://biorxiv.org/content/early/2019/02/14/548628.short
4100  - http://biorxiv.org/content/early/2019/02/14/548628.full
AB  - 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.