AuthorsMueller, Stefan H
Fitschen, Lucy J.
Spenkelink, Lisanne M
van Oijen, Antoine M
KAUST DepartmentBioscience Program
Biological and Environmental Science and Engineering (BESE) Division
Permanent link to this recordhttp://hdl.handle.net/10754/677954
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AbstractThe activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lacking statistical power. We present a highly generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different nucleic-acid enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe a previously unknown activity of the UvrD helicase to remove proteins tightly bound to the ends of DNA.
CitationMueller, S. H., Fitschen, L. J., Shirbini, A., Hamdan, S. M., Spenkelink, L. M., & van Oijen, A. M. (2022). Rapid single-molecule characterisation of nucleic-acid enzymes. https://doi.org/10.1101/2022.03.03.482895
SponsorsThe authors thank Dr Jacob Lewis (University of Wollongong) and Prof. Michael O’Donnell (Rockefeller University) for contributing reagents.
This work was supported by the Australian Research Council (research grants DP150100956 and DP180100858 to A.M.v.O. and an Australian Laureate Fellowship FL140100027 to A.M.v.O.), the National Health and Medical Research Council (NHMRC Investigator grant 2007778 to L.M.S) and an Australian Government Research Training Program Scholarship (to S.H.M). Funding for open access charge: Australian Research Council.
PublisherCold Spring Harbor Laboratory
Except where otherwise noted, this item's license is described as Archived with thanks to Cold Spring Harbor Laboratory, made available under aCC-BY-NC-ND 4.0 International license.