title: Using LacO arrays to monitor DNA double-strand break dynamics in live Schizosaccharomyces pombe cells.
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.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06520, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Leland
authors.forename: Megan C
authors.initials: MC
authors.lastname: King
mesh_terms: D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D053903:DNA Breaks, Double-Stranded; D015870:Gene Expression; D017930:Genes, Reporter; D016681:Genome, Fungal; D042822:Genomic Instability; D059765:Homologous Recombination; D007763:Lac Operon / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D002843:Chromatin