Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation.
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.
Henneke
Ghislaine
G
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Koundrioukoff
Stéphane
S
Hübscher
Ulrich
U
D000255:Adenosine Triphosphate / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000502:physiology*; D045585:Flap Endonucleases; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016196:S Phase
D019925:Cyclin A; D018809:Proliferating Cell Nuclear Antigen; D000255:Adenosine Triphosphate; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human