A Rapidly Inducible DNA Double-Strand Break to Monitor Telomere Formation, DNA Repair, and Checkpoint Activation

The study of processes that govern genome integrity has been augmented by the ability to create a precise DNA double-strand break (DSB) in a short period of time that allows the kinetics of DNA metabolism and protein recruitment to be followed. Defined DSBs are made by expressing endonucleases with long recognition sites that are rare or absent in the genome, and require that the endonuclease is only active when induced. Research in this area in Schizosaccharomyces pombe was limited because rapidly inducible promoters were not available until around 2005, and several rapidly inducible DSB systems are now available. Here, we describe a system to rapidly induce a modified I-SceI endonuclease that can generate a DSB 20 min after induction. I-SceI has no recognition sites in the S. pombe genome, allowing the introduction of complex substrates to monitor the effects of a new DSB in real time. This chapter describes how I-SceI can be most efficiently induced and a simple cell length measurement assay to monitor cell cycle checkpoint activation from a single DSB.