HETEROLOGOUS EXPRESSION OF THE HUMAN CYCLIN-DEPENDENT KINASE INHIBITOR P21(CIP1) IN THE FISSION YEAST, SCHIZOSACCHAROMYCES-POMBE REVEALS A ROLE FOR PCNA IN THE CHK1(-CYCLE CHECKPOINT PATHWAY() CELL)

Fission yeast cells expressing the human gene encoding the cyclin-depe ndent kinase inhibitor protein p21(Cip1) were severely compromised for cell cycle progress. The degree of cell cycle inhibition was related to the level of p21(Cip1) expression. Inhibited cells had a 2C DNA con tent and were judged by cytology and pulsed field gel electrophoresis to be in the G2 phase of the cell cycle. p21 accumulated in the nucleu s and was associated with p34(cdc2) and PCNA. Thus, p21(Cip1) interact s with the same targets in fission yeast as in mammalian cells. Elimin ation of p34(cdc2) binding by mutation within the cyclin-dependent kin ase binding domain of p21(Cip1) exaggerated the cell cycle delay pheno type. By contrast, elimination of PCNA binding by mutation within the PCNA-binding domain completely abolished the cell cycle inhibitory eff ects. Yeast cells expressing wild-type p21(Cip1) and the mutant form t hat is unable to bind p34(cdc2) showed enhanced,sensitivity to UV. Cel l cycle inhibition by p21(Cip1) was largely abolished by deletion of t he chk1(+) gene that monitors radiation damage and was considerably en hanced in cells deleted for the rad3(+) gene that monitors both DNA da mage and the completion of DNA synthesis. Overexpression of PCNA also resulted in cell cycle arrest in G2 and this phenotype was also abolis hed by deletion of chk1(+) and enhanced in cells deleted for rad3(+). These results formally establish a link between PCNA and the products of the rad3(+) and chk1(+) checkpoint genes.