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Xenopus Cdc6 performs separate functions in initiating DNA replication.

Natalya S Frolova·Nancy Schek·Nadia Tikhmyanova·Thomas R Coleman
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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.

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Adenosine TriphosphatasesAdenosine TriphosphateAmino Acid MotifsAnimalsCatalysisCell Cycle ProteinsChromatinChromosomal Proteins, Non-HistoneDNA ReplicationDose-Response Relationship, DrugGenetic VectorsHeLa CellsHumansHydrolysisMicroscopy, FluorescenceModels, BiologicalModels, GeneticNuclear ProteinsProtein BindingProtein ConformationProtein Structure, TertiaryProteinsRecombinant ProteinsTime FactorsXenopusXenopus ProteinsJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

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Natalya S Frolova, Nancy Schek, Nadia Tikhmyanova, Thomas R Coleman. 2002. Xenopus Cdc6 performs separate functions in initiating DNA replication.. https://doi.org/10.1091/mbc.01-08-0382

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