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The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells
U Herbig1, C A Marlar, E Fanning
1Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Molecular Biology of the Cell
|August 6, 1999
Summary
Human Cdc6 (HsCdc6) protein is crucial for DNA replication initiation. Its ability to bind and hydrolyze ATP is essential for this process, as demonstrated by defective mutants inhibiting replication.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Cdc6 protein is vital for initiating DNA replication across various species.
- Understanding the function of human Cdc6 (HsCdc6) is key to comprehending replication control.
Purpose of the Study:
- To clone and characterize the human homologue of Cdc6 (HsCdc6).
- To investigate the role of ATP binding and hydrolysis in HsCdc6 function during DNA replication.
Main Methods:
- Cloning and expression of HsCdc6 as a fusion protein.
- Purification and functional analysis of HsCdc6, including ATP binding and hydrolysis assays.
- Site-directed mutagenesis of Walker A and B motifs.
- Microinjection of wild-type and mutant HsCdc6 into human cells.
Main Results:
- HsCdc6 was successfully expressed and purified in a soluble, active form.
- Purified HsCdc6 specifically bound and slowly hydrolyzed ATP.
- Mutants with altered Walker A or B motifs were defective in ATP hydrolysis but retained binding to HsOrc1 and HsCdc6.
- Mutant HsCdc6 proteins showed aberrant conformations with nucleotides.
- Microinjection of mutant HsCdc6 inhibited DNA replication in human G1 cells.
Conclusions:
- ATP binding and hydrolysis by HsCdc6 are essential for the initiation of DNA replication.
- The study elucidates the critical role of nucleotide interaction in HsCdc6 function.
- HsCdc6 acts as a key regulator in the human DNA replication process.
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