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

Christopher A Belica; Michael A Carpenter; Yanjun Chen; William L Brown; Nicholas H Moeller; Ian T Boylan; Reuben S Harris; Hideki Aihara

doi:10.1016/j.jbc.2024.107410

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

J Biol Chem. 2024 Jun;300(6):107410. doi: 10.1016/j.jbc.2024.107410. Epub 2024 May 23.

Authors

Christopher A Belica¹, Michael A Carpenter², Yanjun Chen³, William L Brown¹, Nicholas H Moeller¹, Ian T Boylan⁴, Reuben S Harris⁵, Hideki Aihara⁶

Affiliations

¹ Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.
² Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas, USA; Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, Texas, USA.
³ Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas, USA.
⁴ Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.
⁵ Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas, USA; Howard Hughes Medical Institute, University of Texas Health San Antonio, San Antonio, Texas, USA. Electronic address: rsh@uthscsa.edu.
⁶ Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA. Electronic address: aihar001@umn.edu.

Abstract

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 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. The assay also offers 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.

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

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

APOBEC Deaminases / metabolism
Cytidine Deaminase* / metabolism
DNA* / chemistry
DNA* / metabolism
Deamination
Enzyme Inhibitors / pharmacology
Humans
Kinetics

Substances

Cytidine Deaminase
DNA
APOBEC Deaminases
APOBEC3 proteins, human
Enzyme Inhibitors

Abstract

Publication types

MeSH terms

Substances

Grants and funding