Methods
In situ assay for analyzing the chromatin binding of proteins in fission yeast
We have developed a simple method to assess chromatin binding of proteins in single fission yeast cells (Schizosaccharomyces pombe). Cells expressing fluorescently tagged proteins are permeabilized by enzymatic digestion and extracted with a detergent-containing buffer, and following fixation are visualized by fluorescence microscopy. This method allows the chromatin association of proteins to be correlated with other cell cycle events without the need for cell synchronization.
Using the DHFR heat-inducible degron for protein inactivation in Schizosaccharomyces pombe
Inactivating a specific protein in vivo can yield important information about its function. One strategy previously developed in Saccharomyces cerevisiae by the Varshavsky group involves fusing a degron, derived from mouse dihydrofolate reductase, to the N-terminus of the target protein, which confers temperature-sensitive ubiquitylation by Ubr1 and hence degradation at the restrictive temperature. We have developed a series of vectors for use of this technique in fission yeast.
Rapid regulation of protein activity in fission yeast
Another method to inactivate proteins conditionally involves fusion of the protein to an estradiol-binding domain. In the absence of estradiol, binding of Hsp90 to the estradiol-binding domain can inactive the protein. We have used this method successfully to regulate subunits of the GINS complex.
Labelling nascent DNA synthesis with EdU
EdU (5-ethynyl-2’-deoxyuridine) can be used like BrdU to label DNA in fission yeast, but the detection is simpler. As with BrdU, cells have to be modified to allow EdU uptake. to label DNA in fission yeast. EdU incorporation activates the DNA-damage checkpoint which is an issue for some experiments.
