Assaying topoisomerase II checkpoints in yeast.
Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations and aberrant DNA condensation during mitosis. Recently, novel top2 alleles in the yeast Saccharomyces cerevisiae revealed a checkpoint control that responds to perturbed Topo II activity. Described in this chapter are protocols for assaying the phenotypes seen in top2 mutants on a cell biological basis in live cells: activation of the Topo II checkpoint using spindle morphology, chromosome condensation using fluorescently labeled chromosomal loci, and cell cycle progression by flow cytometry. Further characterization of this novel checkpoint is warranted so that we can further our understanding of the cell cycle, genomic stability, and the possibility of identifying novel drug targets.
Furniss
Katherine
K
Department of Genetics, University of Minnesota, Minneapolis, MN, USA.
Vas
Amit C J
AC
Lane
Andrew
A
Clarke
Duncan J
DJ
D001681:Biological Assay / Q000379:methods*; D002453:Cell Cycle / Q000502:physiology*; D015825:Chromosomes, Fungal / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D009690:Nucleic Acid Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D004247:DNA; D004250:DNA Topoisomerases, Type II