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Quantifying Activity for Repair of the DNA Lesion 8-Oxoguanine by Oxoguanine Glycosylase 1 (OGG1) in Mouse Adult and Fetal Brain Nuclear Extracts Using Biotin-Labeled DNA

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Developmental Toxicology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1965))

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Abstract

The reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG) is commonly used as a biomarker to measure oxidative stress levels in tissue samples from animals and humans. This lesion also can play a pathogenic role in cancer, birth defects, and neurodegeneration, among other disorders. The level of 8-oxoG may be enhanced due to ROS-initiating environmental factors (e.g., drugs, gamma radiation, microbial infection) or due to a decrease in the activity of oxoguanine glycosylase 1 (OGG1), an enzyme that repairs this lesion. Measurement of the activity of OGG1 can be useful in elucidating mechanisms and complements measurements of 8-oxoG levels in tissues of interest. This protocol describes an assay for measuring the activity of 8-oxoG in mouse adult and fetal brain tissues. Briefly, a synthetic duplex containing the 8-oxoG residue in one of the nucleotides (49-mer), labeled with biotin at the 3′-end, is incubated with protein extract from the tissue of interest containing OGG1, which cleaves the 8-oxoG residue producing a cleavage product of ~27-mer. The percent cleavage quantifies the activity of OGG1 in that tissue. The biotin tag allows rapid and sensitive detection of the cleavage product via chemiluminescence, avoiding the problems of safety and short half-lives of radionuclides encountered in assays employing a radioactively-labeled substrate.

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Acknowledgments

The authors’ research was supported by grants from the Canadian Institutes of Health Research (PJT-156023, MOP-115108, MOP-82812) and the University of Toronto Faculty of Pharmacy.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences and Centre for Pharmaceutical Oncology, University of Toronto, Toronto, ON, Canada

    Shama Bhatia & Peter G. Wells

  2. Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada

    Peter G. Wells

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  1. Shama Bhatia
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  2. Peter G. Wells
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Correspondence to Peter G. Wells .

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Editors and Affiliations

  1. Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT, USA

    Jason M. Hansen

  2. Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

    Louise M. Winn

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Bhatia, S., Wells, P.G. (2019). Quantifying Activity for Repair of the DNA Lesion 8-Oxoguanine by Oxoguanine Glycosylase 1 (OGG1) in Mouse Adult and Fetal Brain Nuclear Extracts Using Biotin-Labeled DNA. In: Hansen, J., Winn, L. (eds) Developmental Toxicology. Methods in Molecular Biology, vol 1965. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9182-2_22

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  • DOI: https://doi.org/10.1007/978-1-4939-9182-2_22

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9181-5

  • Online ISBN: 978-1-4939-9182-2

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