title: The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells.
abstract: The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.
authors.affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
authors.forename: U
authors.initials: U
authors.lastname: Herbig
authors.forename: C A
authors.initials: CA
authors.lastname: Marlar
authors.forename: E
authors.initials: E
authors.lastname: Fanning
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016193:G1 Phase / Q000235:genetics; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D051738:Origin Recognition Complex; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016196:S Phase / Q000235:genetics
chemical_list: C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009711:Nucleotides; D051738:Origin Recognition Complex; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II