OVEREXPRESSION OF THE PROTEIN-KINASE PAK1 SUPPRESSES YEAST DNA-POLYMERASE MUTATIONS

This article presents the identification and characterization of the P AK1 gene of Saccharomyces cerevisiae, and the biochemical characteriza tion of the protein kinase activity that it encodes. Overexpression of the PAKI gene product suppresses temperature-sensitive mutations of t he pol1 (cdc17) gene, which encodes DNA polymerase alpha. Overexpressi on and suppression can be achieved either by expressing PAK1 from a hi gh-copy-number plasmid, or by GAL1-induced transcription of PAK1. Gene disruption of PAK1 indicates that it is not an essential gene. The PA K1 gene encodes a protein with a kinase consensus domain. By deletion analysis and site-directed mutagenesis, we demonstrate that the comple te and active kinase consensus domain is required for suppression. A g lutathione-S-transferase (GST)-Pak1 fusion protein, overproduced in ba cteria, can be purified in an active form with glutathione affinity be ads or by immunoprecipitation. Thus, other protein subunits of Pak1 ar e not required for its activity. In vitro protein kinase assays show t hat GST-Pak1 can autophosphorylate, and can phosphorylate casein as an exogenous substrate. The phenotype of the suppressed cdc17-1 cells in dicates that Pak1 suppression is inefficient and does not restore the wild-type phenotype. Pak1 suppression requires Rad9 function, but Pak1 does not affect Rad9 function. Overexpression of PAK1 does not enhanc e the expression of the POLI gene. Pak1 may function by modifying and partially stabilizing thermolabile DNA polymerases, perhaps during DNA repair, because pak1 mutant cells are caffeine sensitive.