Overexpression of the protein kinase Pak1 suppresses yeast DNA polymerase mutations

• Published in: Molecular & general genetics Vol. 256; no. 1; pp. 45 - 53
• Main Authors: Hovland, P.G, Tecklenberg, M, Sclafani, R.A
• Format: Journal Article
• Language: English
• Published: Germany 01.09.1997
• Subjects: Amino Acid Sequence; amino acid sequences; Base Sequence; Cell Cycle Proteins; Cloning, Molecular; dna ligase; DNA Polymerase I - genetics; DNA-directed DNA polymerase; enzyme activity; Fungal Proteins - genetics; Fungal Proteins - metabolism; genbank/u13398; genbank/u18916; gene expression; Gene Expression Regulation, Fungal - genetics; gene overexpression; Genes, Fungal - genetics; Genotype; Immunoblotting; inhibition; ligases; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; nucleotide sequences; pak1 gene; phosphorylation; Precipitin Tests; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Analysis, DNA; structural genes; Suppression, Genetic
• ISSN: 0026-8925; 1432-1874
• DOI: 10.1007/s004380050544

This article presents the identification and characterization of the PAK1 gene of Saccharomyces cerevisiae, and the biochemical characterization of the protein kinase activity that it encodes. Overexpression of the PAK1 gene product suppresses temperature-sensitive mutations of the poll (cdc 17) gene, which encodes DNA polymerase alpha. Overexpression and suppression can be achieved either by expressing PAK1 from a high-copy-number plasmid, or by GAL1-induced transcription of PAK1. Gene disruption of PAK1 indicates that it is not an essential gene. The PAK1 gene encodes a protein with a kinase consensus domain. By deletion analysis and site-directed mutagenesis, we demonstrate that the complete and active kinase consensus domain is required for suppression. A glutathione-S-transferase (GST)-Pak1 fusion protein, overproduced in bacteria, can be purified in an active form with glutathione affinity beads or by immunoprecipitation. Thus, other protein subunits of Pak1 are not required for its activity. In vitro protein kinase assays show that GST-Pak1 can autophosphorylate, and can phosphorylate casein as an exogenous substrate. The phenotype of the suppressed cdc17-1 cells indicates 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 enhance the expression of the POL1 gene. Pak1 may function by modifying and partially stabilizing thermolabile DNA polymerases, perhaps during DNA repair, because pak1 mutant cells are caffeine sensitive.