Second-site suppressor or CDC24-G1, CSC2-1, is identified as PFH1+

The fission yeast Cdc24 protein is proposed to be required for complete processing of the lagging strand in DNA replication. When this protein is mutated, it results in chromosome breakage and loss of cell viability at the restrictive temperature of 36°C (ts) (Nasmyth and Nurse, 1981; Gould et al., 1998). Previous research showed that, besides DNA replication, the Cdc24 protein also participates in DNA repair, telomere maintenance, and sister chromatid cohesion (Bua, 2006; Gould et al., 1998; Tanaka et al., 1999, 2002; Williams and McIntosh, 2002). However, it is still unknown how this protein works. In 2012, Shani Chapman in the Pasion lab isolated two cold-sensitive mutants carrying second-site suppressors of the cdc24-Gl truncation mutation, cscl-1 and csc2-l (Chapman, 2013). The identity and function of cscl and csc2 genes are still unknown. Using flow cytometry analysis, she showed that the cscl-1 mutant arrests in mitosis at 15°C, which implicated the role of the cscl gene in mitosis. In this study, using random spore analysis, we showed that cscl and csc2 are not the same gene. Flow cytometry analysis and DAPI staining show that the csc2-l mutant also arrests in M phase at 15°C. Genomic sequencing of csc2-l mutant shows that one of the mutations is in the pfhl+ gene. Although pfhl+ does not rescue the cold-sensitive phenotype of the csc2-l mutant at 15°C, the cells are no longer elongated. Furthermore, expression ofpfhl+ in the csc2cdc24 double mutant restores the cdc24 temperature sensitive (ts) lethality at 34°C, which is consistent withpfhl+ rescuing the suppression by csc2-l. In addition, expression ofpflil+ in the csclcdc24 mutant also restores the cdc24 ts lethality at 34°C, suggesting that pflil+ is a multi-copy suppressor of cscl-1.