High-Throughput Exonuclease Assay Based on the Fluorescent Base Analogue 2-Aminopurine

Abstract
Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication, DNA repair, and telomere maintenance. Due to their essential role, they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited. Here, we present a simple and versatile real-time exonuclease assay based on 2-aminopurine, an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA. We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both 3′ and 5′ DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor screening campaign. Using our assay, we discover a novel inhibitor of the Mycobacterium tuberculosis PHP-exonuclease that is part of the replicative DNA polymerase DnaE1. Hence, our novel assay will be a useful tool for high-throughput screening for novel exonuclease inhibitors that may interfere with DNA replication or DNA maintenance.

Citation
Botto, M. M., Murthy, S., & Lamers, M. H. (2023). High-Throughput Exonuclease Assay Based on the Fluorescent Base Analogue 2-Aminopurine. ACS Omega. https://doi.org/10.1021/acsomega.2c06577

Acknowledgements
Pol δ was a kind gift from the laboratory of Prof. Hamdan at King Abdullah University of Science and Technology, Saudi Arabia. This work was supported by a LUMC Research Fellowship to M.H.L.

Publisher
American Chemical Society (ACS)

Journal
ACS Omega

DOI
10.1021/acsomega.2c06577

Additional Links
https://pubs.acs.org/doi/10.1021/acsomega.2c06577

Permanent link to this record