As the cell enters mitosis it is important that DNA replication is complete and that any resulting DNA damage has been removed. Hence specific cell cycle 'checkpoint' mechanisms have evolved to monitor the condition of the genome, allowing the cell to temporarily arrest the cell cycle should DNA replication be incomplete or should the DNA need to be repaired. One point at which checkpoints arrest the cell cycle is before mitosis since the segregation of damaged chromosomes is likely to be deleterious and lead to the accumulation of mutations. In Schizosaccharomyces pombe the G2/M DNA damage checkpoint acts through the Chk1 gene product which is a protein kinase. Ultimately this checkpoint is thought to interact with the ubiquitous p34cdc2 protein, since chk1 was isolated as a supressor of a cold sensitive cdc2 mutation. In order to identify further componants of this pathway a mutant screen was carried out to isolate suppressors of the chk1 overexpression phenotype (elongated cells due to continued growth with a G2 block), that are also radiation sensitive. A plasmid able to rescue one of these mutants, which also mapped to the correct locus, identified a gene thought to function downstream of chk1, which is currently being sequenced. We are investigating Chk1 function by mutational analysis of the kinase domain and of the regions most highly conserved between the S. pombe gene and its S. cerevisiae homologue. Potential interactions with other checkpoint, cdc and suppressor proteins are also being investigated using co-immunoprecipitation and 2-hybrid analyses.