Overexpression of the protein kinase Pak1 suppresses yeast DNA polymerase mutations.
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.
Hovland
P G
PG
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262, USA.
Tecklenberg
M
M
Sclafani
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics; D005838:Genotype; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013489:Suppression, Genetic
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; C094326:PRK1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D004256:DNA Polymerase I