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Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation
Ghislaine Henneke1, Stéphane Koundrioukoff, Ulrich Hübscher
1Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Abstract:
Cyclin-dependent kinase (Cdk) Cdk1-Cyclin A can phosphorylate Flap endonuclease 1 (Fen1), a key-enzyme of the DNA replication machinery, in late S phase. Cdk1-cyclin A forms a complex in vitro and in vivo with Fen1. Furthermore, Fen1 phosphorylation is detected in vivo and depends upon Cdks activity. As a functional consequence of phosphorylation by Cdk1-Cyclin A in vitro, endo- and exonuclease activities of Fen1 are reduced whereas its DNA binding is not affected. Moreover, phosphorylation of Fen1 by Cdk1-Cyclin A abrogates its proliferating cell nuclear antigen (PCNA) binding thus preventing stimulation of Fen1 by PCNA. Concomitantly, human cells expressing the S187A mutant defective for Cdk1-Cyclin A phosphorylation accumulate in S phase consistent with a failure in cell cycle regulation through DNA replication. Our results suggest a novel regulatory role of Cdks onto the end of S phase by targeting directly a key enzyme involved in DNA replication.
Insights
Cyclin-dependent kinases (Cdks) regulate DNA replication by phosphorylating Flap endonuclease 1 (Fen1), reducing its activity and preventing PCNA stimulation. This phosphorylation is crucial for cell cycle progression.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Replication
Background:
- Flap endonuclease 1 (Fen1) is a key enzyme in DNA replication.
- Cyclin-dependent kinases (Cdks) are critical regulators of the cell cycle.
Purpose of the Study:
- To investigate the regulatory role of Cdk1-Cyclin A on Fen1 activity.
- To understand the impact of Fen1 phosphorylation on DNA replication and cell cycle progression.
Main Methods:
- In vitro and in vivo complex formation assays between Cdk1-Cyclin A and Fen1.
- Analysis of Fen1 endonuclease and exonuclease activities.
- Assessment of Fen1-PCNA binding.
- Cell cycle analysis of human cells expressing a phosphorylation-defective Fen1 mutant.
Main Results:
- Cdk1-Cyclin A directly phosphorylates Fen1 in vitro and in vivo.
- Phosphorylation reduces Fen1's endonuclease and exonuclease activities but not DNA binding.
- Phosphorylation abrogates Fen1's interaction with PCNA, inhibiting PCNA-mediated stimulation.
- Human cells with a non-phosphorylatable Fen1 mutant (S187A) arrest in S phase, indicating impaired cell cycle regulation.
Conclusions:
- Cdk1-Cyclin A-mediated phosphorylation of Fen1 represents a novel regulatory mechanism at the end of S phase.
- This regulation impacts DNA replication fidelity and cell cycle progression.
- Targeting Fen1 by Cdks provides a direct link between cell cycle control and DNA replication machinery.