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Monitoring Nuclease Activity by X-Ray Scattering Interferometry Using Gold Nanoparticle-Conjugated DNA

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DNA Damage Responses

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

Abstract

The biologically critical, exquisite specificity and efficiency of nucleases, such as those acting in DNA repair and replication, often emerge in the context of multiple other macromolecules. The evolved complexity also makes biologically relevant nuclease assays challenging and low-throughput. Meiotic recombination 11 homolog 1 (MRE11) is an exemplary nuclease that initiates DNA double-strand break (DSB) repair and processes stalled DNA replication forks. Thus, DNA resection by MRE11 nuclease activity is critical for multiple DSB repair pathways as well as in replication. Traditionally, in vitro nuclease activity of purified enzymes is studied either through gel-based assays or fluorescence-based assays like fluorescence resonance energy transfer (FRET). However, adapting these methods for a high-throughput application such as inhibitor screening can be challenging. Gel-based approaches are slow, and FRET assays can suffer from interference and distance limitations. Here we describe an alternative methodology to monitor nuclease activity by measuring the small-angle X-ray scattering (SAXS) interference pattern from gold nanoparticles (Au NPs) conjugated to 5′-ends of dsDNA using X-ray scattering interferometry (XSI). In addition to reporting on the enzyme activity, XSI can provide insight into DNA-protein interactions, aiding in the development of inhibitors that trap enzymes on the DNA substrate. Enabled by efficient access to synchrotron beamlines, sample preparation, and the feasibility of high-throughput XSI data collection and processing pipelines, this method allows for far greater speeds with less sample consumption than conventional SAXS techniques. The reported metrics and methods can be generalized to monitor not only other nucleases but also most other DNA-protein interactions.

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Acknowledgments

The work is supported by R35 CA220430, by the Cancer Prevention Research Institute of Texas (CPRIT-grant#RP180813), and by a Robert A. Welch Chemistry Chair. Efforts to apply SAXS to characterize eukaryotic pathways relevant to human cancers and merge nano- to mesoscale structures are supported in part by National Cancer Institute grants Structural Biology of DNA Repair (SBDR) CA092584. SAXS data was collected at the Advanced Light Source (ALS) beamline SIBYLS which is supported by the DOE-BER IDAT DE-AC02-05CH11231 and NIGMS ALS-ENABLE (P30 GM124169 and S10OD018483).

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Author notes

  1. Daniel J. Rosenberg and Aleem Syed contributed equally to the work described in this chapter.

Authors and Affiliations

  1. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Daniel J. Rosenberg, John A. Tainer & Greg L. Hura

  2. Graduate Group in Biophysics, University of California, Berkeley, Berkeley, CA, USA

    Daniel J. Rosenberg

  3. Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

    Aleem Syed & John A. Tainer

  4. Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    John A. Tainer

  5. Chemistry and Biochemistry Department, University of California Santa Cruz, Santa Cruz, CA, USA

    Greg L. Hura

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  1. Daniel J. Rosenberg
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  2. Aleem Syed
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  3. John A. Tainer
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  4. Greg L. Hura
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Corresponding authors

Correspondence to John A. Tainer or Greg L. Hura .

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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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Rosenberg, D.J., Syed, A., Tainer, J.A., Hura, G.L. (2022). Monitoring Nuclease Activity by X-Ray Scattering Interferometry Using Gold Nanoparticle-Conjugated DNA. 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_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2063-2_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2062-5

  • Online ISBN: 978-1-0716-2063-2

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