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A more recent version of this article appeared on April 1, 2002
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Submitted on August 2, 2001
Revised on December 10, 2001
Accepted on January 18, 2002
1 Molecular Oncology Program, Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111
* Corresponding author. E-mail address: TR_Coleman{at}fccc.edu.
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. While 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 prior to wild-type Xcdc6, but stimulates DNA replication when added simultaneously with wild-type Xcdc6; 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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