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Analysis of Cdc6 function in the assembly of mammalian prereplication complexes
Jeanette Gowen Cook1, Chi-Hyun Park, Thomas W Burke
1Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Box 3054, Durham, NC 27710, USA.
Summary
Cdc6 protein initiates DNA replication by loading Mcm proteins onto chromatin in mammalian cells. Its ATPase activity is crucial, but phosphorylation by cyclin-dependent kinases is not required for this process.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Eukaryotic DNA replication initiation involves prereplication complex formation with Cdc6 and minichromosome maintenance (Mcm) complexes.
- Functional analysis of replication proteins in intact mammalian cells has been limited, hindering a complete understanding of the process.
Purpose of the Study:
- To investigate the function of Cdc6 in mammalian DNA replication using adenoviral vectors.
- To determine the role of Cdc6 in Mcm complex loading and DNA replication initiation in quiescent mammalian cells.
Main Methods:
- Adenoviral vectors were used to express normal and mutant forms of Cdc6 in quiescent mammalian cells.
- Analysis of Mcm protein association with chromatin and induction of DNA replication under varying cyclin-dependent kinase (cdk) activity levels.
Main Results:
- Cdc6 expression alone induced Mcm protein association with chromatin in quiescent cells with low cdk activity.
- Cdc6 mediated Mcm loading onto chromatin independently of cdk activity in p21-arrested cells.
- Cdc6 synergized with cyclin E/Cdk2, but not cyclin A/Cdk2, to induce DNA replication. Non-phosphorylatable Cdc6 retained function, while mutations in the ATP-binding site impaired Mcm loading and replication.
Conclusions:
- Cdc6 is sufficient to initiate Mcm chromatin association and DNA replication in mammalian cells.
- Cdc6's ATPase activity is essential for Mcm loading and replication, but cdk phosphorylation is not required.
- Specific domains of Cdc6, including the ATPase domain and noncatalytic amino terminus, are critical for its function in DNA replication.