Search

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 which 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.

Two-dimensional agarose gel electrophoresis is the method of choice to identify and quantify all the topological forms DNA molecules can adopt in vivo. Here we describe the materials and protocols needed to analyze catenanes, the natural outcome of DNA replication, in Saccharomyces cerevisiae. We describe the formation of pre-catenanes during replication and how inhibition of topoisomerase 2 leads to the accumulation of intertwined sister duplexes. This knowledge is essential to determine how replication forks blockage or pausing affects the dynamic of DNA topology during replication.

A detailed protocol of molecular dynamics simulations of supercoiled DNA molecules that can be in addition knotted or catenated is described. We also describe how to model ongoing action of DNA gyrase that introduces negative supercoing into DNA molecules. The protocols provide detailed instructions about model parameters, equations of used potentials, simulation, and visualization. Implementation of the model into a frequently used molecular dynamics simulation environment, ESPResSo, is shown step by step.

Agarose gel electrophoresis is by far the most widely used method for characterizing the topological state of DNA molecules. Although this technique has been used for more than 30 years, the physical mechanism underlying the resolution of topological states remains poorly understood. However, electrophoretic methods remain the most robust and precise techniques for determining the local unwinding of DNA induced by the binding of proteins and small-molecule ligands, analyzing conformational transitions in duplex DNA, measuring changes in helical repeat that accompany shifts in environmental conditions, and characterizing knotting and linking in duplex DNA.