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

  • Bryan A. Leland
  • Megan C. KingEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1176)


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.

Key words

LacO/LacI Chromatin dynamics DNA double-strand break Genome instability Homologous recombination Live-cell imaging Genome engineering S. pombe 



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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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Cell BiologyYale University School of MedicineNew HavenUSA

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