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Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage

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Base Excision Repair Pathway

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2701))

Abstract

Eukaryotic DNA exists in chromatin, where the genomic DNA is packaged into a fundamental repeating unit known as the nucleosome. In this chromatin environment, our genomic DNA is constantly under attack by exogenous and endogenous stressors that can lead to DNA damage. Importantly, this DNA damage must be repaired to prevent the accumulation of mutations and ensure normal cellular function. To date, most in-depth biochemical studies of DNA repair proteins have been performed in the context of free duplex DNA. However, chromatin can serve as a barrier that DNA repair enzymes must navigate in order find, access, and process DNA damage in the cell. To facilitate future studies of DNA repair in chromatin, we describe a protocol for generating nucleosome containing site-specific DNA damage that can be utilized for a variety of in vitro applications. This protocol describes several key steps including how to generate damaged DNA oligonucleotides, the expression and purification of recombinant histones, the refolding of histone complexes, and the reconstitution of nucleosomes containing site-specific DNA damage. These methods will enable researchers to generate nucleosomes containing site-specific DNA damage for extensive biochemical and structural studies of DNA repair in the nucleosome.

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Acknowledgements

This research was supported by the National Institute of General Medical Science R35-GM128562 (B.J.R., T.M.W., J.J.S., and B.D.F) and the National Institute of General Medical Science F32-GM140718 (T.M.W.). We also thank Drs. Karolin Luger, Catherine Musselman, and Michael Poirier for the histone plasmids used in this study.

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  1. Benjamin J. Ryan and Tyler M. Weaver contributed equally to this work

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA

    Benjamin J. Ryan, Tyler M. Weaver, Jonah J. Spencer & Bret D. Freudenthal

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  1. Benjamin J. Ryan
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  2. Tyler M. Weaver
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  3. Jonah J. Spencer
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  4. Bret D. Freudenthal
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Correspondence to Bret D. Freudenthal .

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Editors and Affiliations

  1. Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA

    Kishor K. Bhakat

  2. Department of Internal Medicine, The University of Texas Medical Branch, Galveston, TX, USA

    Tapas K. Hazra

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Ryan, B.J., Weaver, T.M., Spencer, J.J., Freudenthal, B.D. (2023). Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage. In: Bhakat, K.K., Hazra, T.K. (eds) Base Excision Repair Pathway. Methods in Molecular Biology, vol 2701. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3373-1_4

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