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Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins
Isabelle Frouin1, Alessandra Montecucco, Giuseppe Biamonti
1Istituto di Genetica Molecolare-CNR, Pavia, Italy.
The EMBO Journal
|May 15, 2002
Summary
This study isolated a replication competent (RC) complex from HeLa cells, revealing its association with cell-cycle regulators and dynamic protein interactions crucial for DNA replication.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- A replication competent (RC) complex containing DNA polymerase alpha, DNA polymerase delta, and replication factor C was previously isolated from calf thymus.
- Understanding the composition and regulation of DNA replication machinery in human cells is crucial for cell cycle control.
Purpose of the Study:
- To isolate and characterize the RC complex from synchronized HeLa cells.
- To investigate the association of the RC complex with cell-cycle regulatory proteins.
- To elucidate the role of the RC complex in DNA synthesis and its regulation during the cell cycle.
Main Methods:
- Isolation of the RC complex from nuclear extracts of synchronized HeLa cells.
- Analysis of associated proteins, including DNA polymerases and cell-cycle regulators (cyclins, Cdks).
- In vivo cross-linking of proteins to DNA to assess chromatin association.
- Assay of kinase and DNA polymerase activities, including switch between distributive and processive synthesis.
Main Results:
- The HeLa cell RC complex contains DNA replication proteins and cell-cycle regulators like cyclin A, cyclin B1, Cdk2, and Cdk1.
- The complex exhibits kinase activity and DNA polymerase activities that switch modes dependent on proliferating cell nuclear antigen.
- In vivo cross-linking confirmed the association of the RC complex with chromatin.
- Cyclin A/Cdk2 associated with the complex during S phase, while cyclin B1/Cdk1 were present in G2/M phase.
Conclusions:
- The RC complex isolated from HeLa cells is associated with key cell-cycle regulatory proteins.
- The dynamic association of cyclins/Cdks with the RC complex suggests cell-cycle-dependent regulation of its activity and localization.
- Specific cyclin/Cdk complexes likely control the function and positioning of the RC complex during DNA replication.
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