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Vol. 13, Issue 4, 1298-1312, April 2002
Molecular Oncology Program, Institute for Cancer Research, Fox
Chase Cancer Center, Philadelphia, Pennsylvania 19111
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.
Corresponding author. E-mail address:
tr_coleman{at}fccc.edu.
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