Analysis of DNA Metabolism in Fission Yeast

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study DNA metabolism, in which the DNA replication and repair mechanisms are evolutionarily conserved. In this introduction we describe a range of methods commonly used to study aspects of DNA metabolism in fission yeast, focusing on approaches used for the analysis of genome stability, DNA replication, and DNA repair. We describe the use of a minichromosome, Ch¹⁶, for monitoring different aspects of genome stability. We introduce two-dimensional gel electrophoresis and immunofluorescent visualization of combed DNA molecules for the analysis of DNA replication. Further, we introduce a pulsed field gel electrophoresis (PFGE) assay to physically monitor chromosome integrity, which can be used in conjunction with a DNA double-strand break (DSB) repair assay to genetically quantitate different DSB repair and misrepair outcomes, including gross chromosomal rearrangements, in fission yeast.