Work

Hijacking a real time detection thermocycler for enzymology: Improvement of a fluorescent bulk assay monitoring helicase activity

Year: 2025

Type: article

Abstract: Helicases are enzymes involved in all aspects of nucleic acid synthesis, regulation and degradation. As a consequence, several methods were developed to monitor their enzymatic activity. In this report, we described an improvement of bulk fluorescent helicase assays to overcome their specific limitations (cost, health and safety regulations, etc.). Using a real time detection thermocycler to monitor the fluorescence in real-time, we managed to precisely control the initiation of the helicase reaction through temperature tuning. Therefore, we were able to demonstrate that this setup could provide a qualitative and a quantitative evaluation of the helicase domain of the UPF1 helicase (UPF1-HD) and that several fluorophores could be used in parallel during the same run.

Source: Biochimie

DOI: 10.1016/j.biochi.2025.02.002

Authors: Jean-Philippe Robin, Vincent Mocquet

Institutions: Lyon 1 Université, École Normale Supérieure de Lyon, Centre National de la Recherche Scientifique, Inserm, Laboratoire de Biologie et Modélisation de la Cellule

Language: English

Topic: DNA Repair Mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,110

Funder: École Normale Supérieure de Lyon