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

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

Department of Biochemistry & Structural Biology

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

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.

Original language	English (US)
Article number	107410
Journal	Journal of Biological Chemistry
Volume	300
Issue number	6
DOIs	https://doi.org/10.1016/j.jbc.2024.107410
State	Published - Jun 2024

Keywords

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

ASJC Scopus subject areas

Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.jbc.2024.107410

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title = "A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination",

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",

author = "Belica, {Christopher A.} and Carpenter, {Michael A.} and Yanjun Chen and Brown, {William L.} and Moeller, {Nicholas H.} and Boylan, {Ian T.} and Harris, {Reuben S.} and Hideki Aihara",

note = "Publisher Copyright: {\textcopyright} 2024 The Authors",

year = "2024",

month = jun,

doi = "10.1016/j.jbc.2024.107410",

language = "English (US)",

volume = "300",

journal = "Journal of Biological Chemistry",

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T1 - A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination

AU - Belica, Christopher A.

AU - Carpenter, Michael A.

AU - Chen, Yanjun

AU - Brown, William L.

AU - Moeller, Nicholas H.

AU - Boylan, Ian T.

AU - Harris, Reuben S.

AU - Aihara, Hideki

PY - 2024/6

Y1 - 2024/6

N2 - 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.

AB - 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.

KW - APOBEC3

KW - biotechnology

KW - cancer

KW - cytidine deaminase

KW - fluorescence resonance energy transfer (FRET)

KW - high-throughput screening (HTS)

KW - mutagenesis

KW - protein-DNA interaction

KW - real-time assay

KW - somatic mutations

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AN - SCOPUS:85195814895

SN - 0021-9258

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JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 6

M1 - 107410

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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