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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / genetics
  • Chromatin / metabolism
  • Chromatin Assembly and Disassembly
  • DNA Breaks, Double-Stranded*
  • Gene Expression
  • Genes, Reporter
  • Genome, Fungal
  • Genomic Instability
  • Homologous Recombination
  • Lac Operon / genetics*
  • Plasmids / genetics
  • Schizosaccharomyces / genetics*
  • Transformation, Genetic

Substances

  • Chromatin