title: Xenopus Cdc6 performs separate functions in initiating DNA replication.
abstract: Cdc6 performs an essential role in the initiation of eukaryotic DNA replication by recruiting the minichromosome maintenance (MCM) complex onto DNA. Using immunodepletion/add-back experiments in Xenopus egg extracts, we have determined that both Walker A (ATP binding) and Walker B (ATP hydrolysis) motifs of Xenopus Cdc6 (Xcdc6) are essential, but have distinct functional roles. Although Walker B mutant protein binds chromatin well, Walker A mutant protein binds chromatin poorly. Neither Walker A nor Walker B mutant protein, however, load appreciable MCM onto DNA. Herein, we provide evidence that Cdc6 functions as a multimer: 1) mutant and wild-type Xcdc6 form multimers; 2) either mutant protein is dominant negative when added before wild-type Xcdc6, but stimulates DNA replication when added simultaneously with wild-type Xcdc6; and 3) the two mutants restore DNA replication when added together, in the absence of wild-type Xcdc6. Our findings suggest that ATP may play a key regulatory role within this multimer: its binding to Cdc6 promotes chromatin association and its hydrolysis facilitates MCM loading. Moreover, ATP binding and hydrolysis may occur in trans between Cdc6 subunits within the complex.
authors.affiliation: Molecular Oncology Program, Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
authors.forename: Natalya S
authors.initials: NS
authors.lastname: Frolova
authors.forename: Nancy
authors.initials: N
authors.lastname: Schek
authors.forename: Nadia
authors.initials: N
authors.lastname: Tikhmyanova
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Coleman
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism* / Q000502:physiology*; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus; D029867:Xenopus Proteins
chemical_list: C103706:CDC6 protein, human; C118388:Cdc6 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases