title: Characterization of a Drosophila ortholog of the Cdc7 kinase: a role for Cdc7 in endoreplication independent of Chiffon.
abstract: Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Robert
authors.initials: R
authors.lastname: Stephenson
authors.affiliation: From the Departments of Biochemistry and.
authors.forename: Marcus R
authors.initials: MR
authors.lastname: Hosler
authors.affiliation: Chemistry and Medicinal Chemistry, and.
authors.forename: Navnath S
authors.initials: NS
authors.lastname: Gavande
authors.affiliation: Chemistry and Medicinal Chemistry, and Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: From the Departments of Biochemistry and Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907 vweake@purdue.edu.
authors.forename: Vikki M
authors.initials: VM
authors.lastname: Weake
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D004261:DNA Replication; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006657:Histones / Q000737:chemistry; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D012568:Schizosaccharomyces; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D029721:Drosophila Proteins; D004527:Egg Proteins; D006657:Histones; D011994:Recombinant Proteins; C121887:chif protein, Drosophila; C000597013:Cdc7 protein, Drosophila; D017346:Protein Serine-Threonine Kinases
keywords: Cdc7; Cell Cycle; DNA Replication; Dbf4; Drosophila; Endoreplication; Gene Amplification; Phosphorylation; Serine-Threonine Protein Kinase