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-exonuclea

SUBMITTER: Botto MM 

PROVIDER: S-EPMC9996622 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature