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In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing

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DNA Damage Responses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2444))

Abstract

RAD51-mediated homologous recombination (HR) is a conserved mechanism for the repair of DNA double-strand breaks and the maintenance of DNA replication forks. Several breast and ovarian tumor suppressors, including BRCA1 and BARD1, have been implicated in HR since their discovery in the 1990s. However, a holistic understanding of how they participate in HR has been hampered by the immense challenge of expressing and purifying these large and unstable protein complexes for mechanistic analysis. Recently, we have overcome such a challenge for the BRCA1-BARD1 complex, allowing us to demonstrate its pivotal role in HR via the promotion of RAD51-mediated DNA strand invasion. In this chapter, we describe detailed procedures for the expression and purification of the BRCA1-BARD1 complex and in vitro assays using this tumor suppressor complex to examine its ability to promote RAD51-mediated homologous DNA pairing. This includes two distinct biochemical assays, namely, D-loop formation and synaptic complex assembly. These methods are invaluable for studying the BRCA1-BARD1 complex and its functional interplay with other factors in the HR process.

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Acknowledgments

We thank members of the Zhao laboratory (O’Taveon Fitzgerald, Wenjing Li and Yuxin Huang) for critical reading on the manuscript and Dr. Jeffrey D. Parvin for providing the pFastBac-Flag-BRCA1 and pFastbac-BARD1 plasmids. This work was supported by foundation grants (V Scholar V2019.Q13 from V Foundation for Cancer Research and Young Investigator Award from Max and Minnie Tomerlin Voelcker Fund) and startup funds from University of Texas Health Science Center at San Antonio (all to W.Z.). C.M.R. is supported by a T32 (AG 021890) through the UT Health San Antonio Biology of Aging program.

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Authors and Affiliations

  1. Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Meiling Wang, Cody M. Rogers, Dauren Alimbetov & Weixing Zhao

Authors
  1. Meiling Wang
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  2. Cody M. Rogers
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  3. Dauren Alimbetov
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  4. Weixing Zhao
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Corresponding author

Correspondence to Weixing Zhao .

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Editors and Affiliations

  1. Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA

    Nima Mosammaparast

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Wang, M., Rogers, C.M., Alimbetov, D., Zhao, W. (2022). In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing. In: Mosammaparast, N. (eds) DNA Damage Responses. Methods in Molecular Biology, vol 2444. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2063-2_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2063-2_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2062-5

  • Online ISBN: 978-1-0716-2063-2

  • eBook Packages: Springer Protocols

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