title: Drf1-dependent kinase interacts with Claspin through a conserved protein motif.
abstract: The Dbf4/Drf1-dependent kinase (DDK) is required for the initiation of DNA replication in eukaryotes. Another protein, Claspin, mediates the activation of a cellular checkpoint response to stalled replication forks and is also a regulator of replication. In this study, we found that DDK phosphorylates Claspin in vitro and forms a nuclear complex containing Cdc7, Drf1, and Claspin in Xenopus egg extracts. In addition, purified Claspin and DDK are capable of a direct in vitro interaction. We identified a conserved binding site on Claspin required for its interaction with DDK. This site corresponds to the first of two sequence repeats in the Chk1-binding domain of Claspin. Furthermore, we have established that two amino acids in this motif, Asp(861) and Gln(866), are essential for the interaction between Claspin and DDK. We found that mutant forms of Claspin incapable of interacting with DDK are still able to associate with and activate Chk1 in response to DNA replication blockages. However, Claspin-depleted egg extracts that have been reconstituted with these mutants of Claspin undergo DNA replication more slowly. These findings suggest that the interaction of DDK with Claspin mediates a checkpoint-independent function of Claspin related to DNA replication.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Gold
authors.forename: William G
authors.initials: WG
authors.lastname: Dunphy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D000080865:Formins; D006801:Humans; D009154:Mutation; D010063:Ovum / Q000201:enzymology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C416368:CLSPN protein, Xenopus; C488158:CLSPN protein, human; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C505573:DBF4B protein, Xenopus; C108962:DIAPH1 protein, human; C505572:Dbf4 protein, Xenopus; D000080865:Formins; D029867:Xenopus Proteins; D011494:Protein Kinases; C489405:CDC7 protein, Xenopus; C068619:CDC7 protein, human; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases