Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase

Dfp1/Him1 protein of fission yeast, Schizosaccharomyces pombe, encodes the regulatory subunit for Hsk1 kinase, a homologue of budding yeast Cdc7 kinas e essential for initiation and progression of the S phase of the cell cycle . This protein binds and activates Hsk1 kinase, which phosphorylates the MC M2 protein. Comparison of the amino acid sequences of the Cdc7 regulatory s ubunits from various eukaryotes revealed the presence of three small stretc hes of conserved amino acid sequences, namely Dbf4 motifs N, M, and C. We r eport here that the Dbf4 motif M, a unique proline-rich motif, and the Dbf4 motif C, a C2H2-type zinc finger motif, are essential for mitotic function s of Dfp1/Him1 protein as well as for full-level activation of Hsk1 kinase. In vitro, a small segment containing the Dbf4 motif M or C alone binds to and partially activates Hsk1. Co-expression of these two segments augments the extent of activation. Furthermore, a fused polypeptide containing only Dbf4 motifs M and C without any spacer can activate Hsk1 and is capable of rescuing the growth defect of him1 null cells. Insertion of a long stretch of amino acids between the motif M and motif C can be tolerated for mitotic functions. On the other hand, internal deletion of Dbf4 motif N, which has some similarity with the BRCA C-terminal domain motif, results in a defect in hydroxyurea-induced checkpoint responses and sensitivity to methyl meth ane sulfonate, yet mitotic functions and kinase activation are intact. In o ne-hybrid assays with budding yeast Dbf4, motif N mutants exhibit reduced i nteraction with a replication origin. Our observations suggest the molecula r architecture of Cdc7-Dbf4-related kinase complexes at the origins, in whi ch they are tethered to replication machinery through Dbf4 motif N and the catalytic subunits are activated through bipartite binding of Dbf4 motifs M and C of the regulatory subunits.