A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination.

APOBEC3 biotechnology cancer cytidine deaminase fluorescence resonance energy transfer (FRET) high‐throughput screening (HTS) mutagenesis protein‐DNA interaction real-time assay somatic mutations

Journal

The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R

Informations de publication

Date de publication:
23 May 2024
Historique:
received: 11 01 2024
revised: 10 05 2024
accepted: 12 05 2024
medline: 26 5 2024
pubmed: 26 5 2024
entrez: 25 5 2024
Statut: aheadofprint

Résumé

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics and also offering a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Identifiants

DOI: 10.1016/j.jbc.2024.107410 PMID: 38796062
pubmed: 38796062
pii: S0021-9258(24)01911-2
doi: 10.1016/j.jbc.2024.107410
pii:
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

107410

Informations de copyright

Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Auteurs

Christopher A Belica (CA)

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA, 55455; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, USA.

Michael A Carpenter (MA)

Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas, 78229, USA; Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, Texas, 78229, USA.

Yanjun Chen (Y)

Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas, 78229, USA.

William L Brown (WL)

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA, 55455; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, USA.

Nicholas H Moeller (NH)

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA, 55455; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, USA.

Ian T Boylan (IT)

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA, 55455; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, USA.

Reuben S Harris (RS)

Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas, 78229, USA; Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, Texas, 78229, USA. Electronic address: rsh@uthscsa.edu.

Hideki Aihara (H)

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA, 55455; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, USA. Electronic address: aihar001@umn.edu.

Classifications MeSH