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DNA unwinding is an Mcm complex-dependent and ATP hydrolysis-dependent process
David Shechter1, Carol Y Ying, Jean Gautier
1Integrated Program in Cellular, Molecular, and Biophysical Studies, Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
The Journal of Biological Chemistry
|August 25, 2004
Summary
Minichromosome maintenance (Mcm) proteins are crucial for DNA replication initiation. Studies show Mcm proteins function as the replicative helicase, unwinding DNA after replication begins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Minichromosome maintenance (Mcm) proteins are essential for DNA replication in eukaryotes.
- Mcm proteins possess conserved helicase motifs and in vitro DNA helicase/ATP hydrolysis activities.
- Their precise role during DNA replication elongation, particularly as the replicative helicase, remains unclear.
Purpose of the Study:
- To investigate the function of Mcm proteins during DNA replication elongation.
- To determine if Mcm proteins act as the replicative helicase responsible for DNA unwinding at the replication fork.
Main Methods:
- Utilized Xenopus laevis egg nucleoplasmic extract (NPE).
- Employed purified polyclonal antibodies against six Xenopus Mcm proteins (Mcm2-7).
- Performed quantitative depletion experiments and antibody-mediated inhibition assays.
- Used p21(cip1) inhibitor and ATPgammaS to dissect origin firing and helicase function.
Main Results:
- Mcm proteins are required for DNA replication and unwinding post-initiation.
- Depletion of Mcms did not affect replication, indicating their role before pre-replicative complex assembly.
- Antibody inhibition blocked replication and unwinding, confirming Mcm requirement.
- Plasmid unwinding was dependent on ATP hydrolysis, supporting helicase function.
Conclusions:
- The Mcm protein complex functions as the replicative helicase.
- Mcm proteins are essential for DNA unwinding during replication elongation.
- Their activity is ATP hydrolysis-dependent.