Constructing a Thermosensitive Fission Yeast Mutant to Monitor Cdc24 Localization During the Cell Cycle

Maintaining genome integrity is critical for the survival of organisms. Genome instability may arise due to mutations in genes that are involved in DNA replication or repair. Fission yeast cells with mutations in the cdc24⁺ gene arrest in S-phase of the cell cycle and exhibit chromosome breaks. Although the precise role of cdc24 is unknown, the Cdc24 protein has been suggested to play a role in lagging strand synthesis based upon genetic and physical interactions with conserved replication proteins that are know to be chromatin-associated.  My main goal is to determine if Cdc24 is bound to chromatin by performing a chromatin fractionation assay.  Although Cdc24 is a nuclear protein throughout the cell cycle, it may exhibit cell cycle-regulated chromatin association. Here we report the construction of a thermosensitive cdc10 strain carrying a HA-epitope tagged cdc24 allele by random spore analysis.  cdc10 mutants arrest in G1 at their restrictive temperature and release into a synchronous progression at the permissive temperature.  The measure of synchrony and cell cycle progression for this thermosensitive mutant will be monitored by flow cytometry (DNA content) and microscopy (septation index). This approach allows us to monitor Cdc24 chromatin association at different phases of the cell cycle. Results from these experiments will help further define a model for Cdc24 function.