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Single-Cell Analysis of Histone Acetylation Dynamics at Replication Forks Using PLA and SIRF

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HDAC/HAT Function Assessment and Inhibitor Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2589))

Abstract

Genome integrity is constantly challenged by various processes including DNA damage, structured DNA, transcription, and DNA-protein crosslinks. During DNA replication, active replication forks that encounter these obstacles can result in their stalling and collapse. Accurate DNA replication requires the ability of forks to navigate these threats, which is aided by DNA repair proteins. Histone acetylation participates in this process through an ability to signal and recruit proteins to regions of replicating DNA. For example, the histone acetyltransferase PCAF promotes the recruitment of the DNA repair factors MRE11 and EXO1 to stalled forks by acetylating histone H4 at lysine 8 (H4K8ac). These highly dynamic processes can be detected and analyzed using a modified proximity ligation assay (PLA) method, known as SIRF (in situ protein interactions with nascent DNA replication forks). This single-cell assay combines PLA with EdU-coupled Click-iT chemistry reactions and fluorescence microscopy to detect these interactions at sites of replicating DNA. Here we provide a detailed protocol utilizing SIRF that detects the HAT PCAF and histone acetylation at replication forks. This technique provides a robust methodology to determine protein recruitment and modifications at the replication fork with single-cell resolution.

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Acknowledgments

This work in the K.M.M. laboratory was supported by the National Institutes of Health, National Cancer Institute (R01CA198279 and R01CA201268). The J.K. laboratory was supported by the National Research Foundation of Korea (NRF-2022R1C1C1007759).

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Authors and Affiliations

  1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA

    Seo Yun Lee, Jae Jin Kim & Kyle M. Miller

  2. Department of Life Science and Multidisciplinary Genome Institute, Hallym University, Chuncheon, Republic of Korea

    Jae Jin Kim

  3. Livestrong Cancer Institutes, Dell Medical School, The University of Texas at Austin, Austin, TX, USA

    Kyle M. Miller

Authors
  1. Seo Yun Lee
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  2. Jae Jin Kim
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Corresponding authors

Correspondence to Jae Jin Kim or Kyle M. Miller .

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Editors and Affiliations

  1. Institute of Toxicology, University Medical Center Mainz, Mainz, Germany

    Oliver H. Krämer

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Lee, S.Y., Kim, J.J., Miller, K.M. (2023). Single-Cell Analysis of Histone Acetylation Dynamics at Replication Forks Using PLA and SIRF. In: Krämer, O.H. (eds) HDAC/HAT Function Assessment and Inhibitor Development. Methods in Molecular Biology, vol 2589. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2788-4_23

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  • DOI: https://doi.org/10.1007/978-1-0716-2788-4_23

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2787-7

  • Online ISBN: 978-1-0716-2788-4

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