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Methods Mol Biol. 2014;1176:127-41. doi: 10.1007/978-1-4939-0992-6_11.

Using LacO arrays to monitor DNA double-strand break dynamics in live Schizosaccharomyces pombe cells.

Author information

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

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.

PMID:
25030924
DOI:
10.1007/978-1-4939-0992-6_11
[Indexed for MEDLINE]

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