title: Protein phosphatase 2A and Cdc7 kinase regulate the DNA unwinding element-binding protein in replication initiation.
abstract: The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2-7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2-7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435.
authors.forename: Yanzhe
authors.initials: Y
authors.lastname: Gao
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435.
authors.forename: Jianhong
authors.initials: J
authors.lastname: Yao
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435.
authors.forename: Sumeet
authors.initials: S
authors.lastname: Poudel
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435.
authors.forename: Eric
authors.initials: E
authors.lastname: Romer
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435.
authors.forename: Lubna
authors.initials: L
authors.lastname: Abu-Niaaj
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435 Michael.Leffak@wright.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Leffak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D054648:Protein Phosphatase 2 / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C500359:DTD1 protein, human; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; D054648:Protein Phosphatase 2; D064110:Minichromosome Maintenance Proteins
keywords: Cdc7; Cell Cycle; Cell Division; DNA Replication; DNA Synthesis; DUE-B; MCM; Protein Phosphatase 2 (PP2A); cdc45