RT Journal Article
SR Electronic
T1 A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 548628
DO 10.1101/548628
A1 Emily C. Sheppard
A1 Sally Rogers
A1 Nicholas J. Harmer
A1 Richard Chahwan
YR 2019
UL http://biorxiv.org/content/early/2019/02/14/548628.abstract
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.