TY - JOUR
T1 - Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism
AU - Mueller, Stefan H
AU - Fitschen, Lucy J
AU - Shirbini, Afnan
AU - Hamdan, Samir
AU - Spenkelink, Lisanne M
AU - van Oijen, Antoine M
N1 - KAUST Repository Item: Exported on 2022-11-04
Acknowledgements: Australian Research Council [DP150100956, DP180100858 to A.M.v.O.]; Australian Laureate Fellowship [FL140100027 to A.M.v.O.]; National Health and Medical Research Council [NHMRC Investigator grant 2007778 to L.M.S.]; Australian Government Research Training Program Scholarship (to S.H.M.). Funding for open access charge: Australian Research Council. The authors thank Dr Jacob Lewis (University of Wollongong) and Prof. Michael O’Donnell (Rockefeller University) for contributing reagents.
PY - 2022/11/2
Y1 - 2022/11/2
N2 - The 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 lack 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 enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5′-, but not 3′-ends of biotinylated DNA.
AB - The 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 lack 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 enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5′-, but not 3′-ends of biotinylated DNA.
UR - http://hdl.handle.net/10754/685394
UR - https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkac949/6793808
U2 - 10.1093/nar/gkac949
DO - 10.1093/nar/gkac949
M3 - Article
C2 - 36321650
SN - 0305-1048
JO - Nucleic Acids Research
JF - Nucleic Acids Research
ER -