Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast.
Members of the Cdc7 family of protein kinases are essential for the initiation of DNA replication in all eukaryotes, but their precise biochemical function is unclear. We have purified the fission yeast Cdc7 homologue Hsk1 approximately 30,000-fold, to near homogeneity. Purified Hsk1 has protein kinase activity on several substrates and is capable of autophosphorylation. Point mutations in highly conserved regions of Hsk1 inactivate the kinase in vitro and in vivo. Overproduction of two of the mutant hsk1 alleles blocks initiation of DNA replication and deranges the mitotic checkpoint, a phenotype consistent with a role for Hsk1 in the early stages of initiation. The purified Hsk1 kinase can be separated into two active forms, a Hsk1 monomer and a heterodimer consisting of Hsk1 complexed with a co-purifying polypeptide, Dfp1. Association with Dfp1 stimulates phosphorylation of exogenous substrates but has little effect on autokinase activity. We have identified Dfp1 as the fission yeast homologue of budding yeast Dbf4. Purified Hsk1 phosphorylates the Cdc19 (Mcm2) subunit of the six-member minichromosome maintenance protein complex purified from fission yeast. Since minichromosome maintenance proteins have been implicated in the initiation of DNA replication, the essential function of Hsk1 at the G1/S transition may be mediated by phosphorylation of Cdc19. Furthermore, the phosphorylation of critical substrates by Hsk1 kinase is likely regulated by association with a Dbf4-like co-factor.
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
G W
GW
Brown
T J
TJ
Kelly
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D004261:DNA Replication; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases; D016196:S Phase; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C094761:HSK1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; C085710:mcm2 protein, S pombe