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Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage

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Poly(ADP-Ribose) Polymerase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2609))

Abstract

Poly(ADP-ribose) (PAR), catalyzed by members of the poly(ADP-ribose) polymerase family of enzymes, is a posttranslational modification with a critical role in most mechanisms of DNA repair. Upon activation of poly(ADP-ribose) polymerase isoforms 1 and 2 (PARP-1 and PARP-2), the proteins of the base excision repair (BER) and single-strand break repair (SSBR) pathways form DNA lesion-dependent, transient complexes to facilitate repair. PAR is central to the temporal dynamics of BER/SSBR complex assembly and disassembly. To enhance cellular PAR analysis, we developed LivePAR, a fluorescently tagged PAR-binding fusion protein and genetically encoded imaging probe for live cell, quantitative analysis of PAR in mammalian cells. LivePAR has the advantage that it enables real-time imaging of PAR formation in cells and significantly overcomes limitations of immunocytochemistry for PAR analysis. This chapter describes the protocols needed to develop cells expressing LivePAR or EGFP-tagged BER proteins and to evaluate laser-induced formation of PAR and comparison to the assembly of the BER proteins XRCC1 and DNA polymerase-β.

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Acknowledgments

RWS is an Abraham A. Mitchell Distinguished Investigator. Research in the Sobol lab on DNA repair, the analysis of DNA damage, and the impact of genotoxic exposure is funded by grants from the National Institutes of Health (NIH) [CA148629, ES014811, ES029518, ES028949, and CA238061], from the National Science Foundation (NSF) [NSF-1841811], and a grant from the DOD [GRANT11998991, DURIP-Navy]. Support is also provided from the Abraham A. Mitchell Distinguished Investigator Fund and from the Mitchell Cancer Institute Molecular & Metabolic Oncology Program Development fund (to RWS). The support for the development of LivePAR was also provided by the Mitchell Cancer Institute Junior Faculty Award (to CAK). Many thanks to Alison Beiser for kee** all our lab reagents in order.

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Author notes

  1. Rasha Q. Al-Rahahleh

    Present address: Department of Pathology and Laboratory Medicine & Legorreta Cancer Center, Brown University, Providence, RI, USA

Authors and Affiliations

  1. Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA

    Christopher A. Koczor, Kate M. Saville, Rasha Q. Al-Rahahleh, Joel F. Andrews, Jianfeng Li & Robert W. Sobol

  2. Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL, USA

    Christopher A. Koczor, Kate M. Saville, Rasha Q. Al-Rahahleh, Jianfeng Li & Robert W. Sobol

  3. Department of Pathology and Laboratory Medicine & Legorreta Cancer Center, Brown University, Providence, RI, USA

    Robert W. Sobol

Authors
  1. Christopher A. Koczor
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  2. Kate M. Saville
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  3. Rasha Q. Al-Rahahleh
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  4. Joel F. Andrews
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  5. Jianfeng Li
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  6. Robert W. Sobol
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Corresponding author

Correspondence to Robert W. Sobol .

Editor information

Editors and Affiliations

  1. Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND, USA

    Alexei V. Tulin

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Koczor, C.A., Saville, K.M., Al-Rahahleh, R.Q., Andrews, J.F., Li, J., Sobol, R.W. (2023). Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage. In: Tulin, A.V. (eds) Poly(ADP-Ribose) Polymerase. Methods in Molecular Biology, vol 2609. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2891-1_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2891-1_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2890-4

  • Online ISBN: 978-1-0716-2891-1

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