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Live-Cell Fluorescence Imaging of RecN in Caulobacter crescentus Under DNA Damage

Part of the Methods in Molecular Biology book series (MIMB,volume 2004)

Abstract

Structural maintenance of chromosomes (SMC) proteins play a central role in the organization, segregation and maintenance of chromosomes across domains of life. In bacteria, an SMC-family protein, RecN, has been implicated to have important functions in DNA damage repair. Recent studies have suggested that RecN is required to increase chromosome cohesion in response to DNA damage and may also stimulate specific events during recombination-based repair. While biochemical and genetic assays provide insights into mechanism of action of RecN and other repair factors, in vivo understanding of activity and regulation of proteins can be predominantly gained via microscopy-based approaches. Here, we describe a protocol to study the localization of fluorescently tagged RecN to a site-specific double-strand break (DSB) in Caulobacter crescentus. We further outline a method to probe RecN dynamics in cells with a single, nonreplicating chromosome. This technique can be used to study the early steps of recombination-based repair and understand the regulation of protein recruitment to and further association with sites of damage.

Key words

  • SMC proteins
  • RecN
  • Caulobacter crescentus
  • Double-strand break repair
  • Microscopy
  • Fluorescence imaging
  • Bacterial DNA damage

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Acknowledgments

We thank Dr. Asha Mary Joseph and other members of the AB lab for comments on the manuscript. AB acknowledges funding from the Tata Institute of Fundamental Research and a Career Development Award from the Human Frontier of Sciences Program.

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Correspondence to Anjana Badrinarayanan .

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Chimthanawala, A., Badrinarayanan, A. (2019). Live-Cell Fluorescence Imaging of RecN in Caulobacter crescentus Under DNA Damage. In: Badrinarayanan, A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_18

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  • DOI: https://doi.org/10.1007/978-1-4939-9520-2_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9519-6

  • Online ISBN: 978-1-4939-9520-2

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