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Using LacO Arrays to Monitor DNA Double-Strand Break Dynamics in Live Schizosaccharomyces pombe Cells

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Cancer Genomics and Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1176))

Abstract

LacO arrays, when combined with LacI-GFP, have been a valuable tool for studying nuclear architecture and chromatin dynamics. Here, we outline an experimental approach to employ the LacO/LacI-GFP system in S. pombe to assess DNA double-strand break (DSB) dynamics and the contribution of chromatin state to DSB repair. Previously, integration of long, highly repetitive LacO arrays in S. pombe has been a challenge. To address this problem, we have developed a novel approach, based on the principles used for homologous recombination-based genome engineering in higher eukaryotes, to integrate long, repetitive LacO arrays with targeting efficiencies as high as 70 %. Combining this facile LacO/LacI-GFP system with a site-specific, inducible DSB provides a means to monitor DSB dynamics at engineered sites within the genome.

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Acknowledgements

We would like to thank the Gasser lab and Russell labs for providing plasmids. This work was supported by the G. Harold and Leila Y. Mathers Charitable Foundation and the Searle Scholar Program (to M.C.K) and an NIGMS training grant T32GM007223 (to B.A.L.).

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Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06520, USA

    Bryan A. Leland & Megan C. King

Authors
  1. Bryan A. Leland
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  2. Megan C. King
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Corresponding author

Correspondence to Megan C. King .

Editor information

Editors and Affiliations

  1. Department of Pathology& Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA

    Narendra Wajapeyee

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Leland, B.A., King, M.C. (2014). Using LacO Arrays to Monitor DNA Double-Strand Break Dynamics in Live Schizosaccharomyces pombe Cells. In: Wajapeyee, N. (eds) Cancer Genomics and Proteomics. Methods in Molecular Biology, vol 1176. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0992-6_11

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  • DOI: https://doi.org/10.1007/978-1-4939-0992-6_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0991-9

  • Online ISBN: 978-1-4939-0992-6

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