Abstract
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.
Highlights
The initiation of DNA replication at eukaryotic chromosomal origins is generally considered to be a two-stage process
Members of the Cdc7 family of protein kinases are absolutely required for the initiation of DNA replication and are highly conserved throughout Eukaryota
We have purified the S. pombe Cdc7 homologue, Hsk1, to apparent homogeneity. This is the first description of the purification of a Cdc7 kinase from any source
Summary
The initiation of DNA replication at eukaryotic chromosomal origins is generally considered to be a two-stage process (reviewed in Refs. 1 and 2). Expression of DBF4 is required at the G1/S transition for initiation of DNA replication [16] and for Cdc kinase activity [12]. The MCM family has six members, all of which are essential proteins that function in DNA replication (reviewed in Ref. 20) These proteins form heteromeric complexes that interact with chromatin and replication origins in vivo [5,6,7, 21, 22]. Budding yeast Cdc7/Dbf expressed in insect cells phosphorylates GST-Mcm2, -Mcm3, -Mcm, and -Mcm fusion proteins in vitro [27] These data are consistent with the possibility that Cdc kinase may phosphorylate one or more MCM subunits at G1/S, heteromeric MCM complexes have not yet been directly tested as substrates.
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