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A Chromatin Fiber Analysis Pipeline to Model DNA Synthesis and Structures in Fission Yeast

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Genome Instability

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

Abstract

Chromatin fibers, first described by Jackson and Pombo (J Cell Biol 140(6):1285–1295, 1998) are prepared from cells lysed on glass coverslips, and require minimal equipment to produce. Since the DNA is not previously treated with denaturing agents, proteins are left intact and may be used to model other DNA-based processes. Such an analysis can be daunting, without a rigorous method for analysis. We describe a pipeline for chromatin fiber use to model DNA replication complexes. Full protocols for chromatin fiber preparation and staining are presented. Further, we have developed an analysis algorithm for One Dimensional Data—Boolean Logic Operations Binning System (ODD-BLOBS). This freely available software defines replication and protein tracts, measures their lengths, and then correlates replicated areas with protein distributions. Our methods and analysis are tested in Schizosaccharomyces pombe (fission yeast) but may be applied to model replication structures across multiple organisms.

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Acknowledgments

We wish to thank Susan Forsburg (University of Southern California, USA) for her support of this project and for providing strain FY3841. The Sabatinos lab is supported by NSERC DG RGPIN/04405-2015.

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Authors and Affiliations

  1. Ryerson University, 350 Victoria Street, Toronto, ON, Canada, M5B 2K3

    Sarah A. Sabatinos

  2. Royal Ontario Museum, 100 Queen’s Park, Toronto, ON, Canada, M5S 2C6

    Marc D. Green

Authors
  1. Sarah A. Sabatinos
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  2. Marc D. Green
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Corresponding author

Correspondence to Sarah A. Sabatinos .

Editor information

Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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Sabatinos, S.A., Green, M.D. (2018). A Chromatin Fiber Analysis Pipeline to Model DNA Synthesis and Structures in Fission Yeast. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_34

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_34

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

  • eBook Packages: Springer Protocols

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