Microscopy techniques to examine dna replication in fission yeast

Abstract

Temporal and spatial visualization of replication proteins and associated structures within the narrow confines of a yeast nucleus is technically challenging. Choosing the appropriate method depends upon the parameters of the experiment, the nature of the molecules to be observed, and the hypothesis to be tested. In this chapter, we review three broad types of visualization: whole-cell fluorescence or immunofluorescence, which is useful for questions of timing and chromatin association; nuclear spreads, which provide greater resolution within the chromatin for co-localization and region-specific effects; and chromatin fibers, which allow observation of labeled proteins and newly synthesized DNA on a linear chromosome. We also suggest a mounting procedure for live fission yeast with fluorescent proteins. We discuss applications of these protocols and some considerations for choosing methods and fluorophores.