Onne-Andrea1, Malik Kahli2,w, Francisca Mechali1, Jean-Marc Lemaitre2, Guillaume Bossis3 Olivier CouxThe Tetradecyltrimethylammonium Biological

Onne-Andrea1, Malik Kahli2,w, Francisca Mechali1, Jean-Marc Lemaitre2, Guillaume Bossis3 Olivier CouxThe Tetradecyltrimethylammonium Biological Activity compact ubiquitin-like modifier (SUMO) pathway is essential for the maintenance of genome stability. We investigated its possible involvement inside the manage of DNA replication during S phase by utilizing the Xenopus cell-free program. Here we show that the SUMO pathway is important to limit the number and, thus, the density of replication origins that are activated in early S phase. We identified cyclin E, which regulates cyclin-dependent kinase 2 (Cdk2) to trigger origin firing, as an S-phase substrate of this pathway. We show that cyclin E is dynamically and extremely conjugated to SUMO2/3 on chromatin, independently of Cdk2 activity and origin activation. Moreover, cyclin E is the predominant SUMO2/3 target on chromatin in early S phase, as cyclin E depletion abolishes, though its readdition restores, the SUMO2/3 signal. Collectively, our data indicate that cyclin E SUMOylation is vital for controlling origin firing after the cyclin E dk2 complex is recruited onto replication origins.de Recherche de Biochimie Macromoleculaire (CRBM), CNRS UMR5237, University Montpellier I and II, 1919 route de Mende, 34293 Montpellier Cedex 05, France. two Institut de Genomique Fonctionnelle (IGF), CNRS UMR5203, University Montpellier I and II, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France. 3 Institut de Genetique Moleculaire Montpellier (IGMM), CNRS UMR5535, University Montpellier I and II, 1919 route de Mende, 34293 Montpellier Cedex 05, France. w Present address: Institut de Biologie de l’Ecole Normale Superieure (IBENS), CNRS UMR8197, Inserm U1024, 46 rue d’Ulm, 75230 Paris Cedex 05, France. Correspondence and requests for components ought to be addressed to C.B.-A. (e-mail: [email protected]).NATURE COMMUNICATIONS | 4:1850 | DOI: 10.1038/ncomms2875 | nature.com/naturecommunications1 Centre2013 Macmillan Publishers Limited. All rights reserved.ARTICLEost-translational modifiers in the little ubiquitin-like modifier (SUMO) loved ones have emerged as important regulators of protein function and fate. SUMOylation , that is the covalent and reversible conjugation of SUMO to target proteins, is crucial for development, division and upkeep of genome stability from yeast to mammals. Among the numerous functions of SUMO modification are regulation of transcription, DNA repair, nuclear transport and formation of sub-nuclear structures1. 3 SUMO isoforms (B100 amino-acid proteins) are expressed in vertebrates: SUMO1, SUMO2 and SUMO3. SUMO2 and three are hugely related and each include a SUMO consensus modification motif that makes it possible for the formation of polySUMO chains, and is absent in SUMO1. SUMOylation occurs through a biochemical pathway that may be analogous to the ubiquitylation cascade, but using a distinct set of enzymes: the E1 SUMO-activating enzyme (SAE1/SAE2), the E2-conjugating enzyme (Ubc9) and, at the very least in some instances, further E3 ligases. The very first evidence of a connection between SUMO and DNA replication and repair came from the discovery that proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity factor, is often conjugated with SUMO at the replication fork9. PCNA SUMOylation has been reported in yeast, Xenopus and recently in mammalian cells, and it appears to happen in the course of S phase under physiological conditions91. On the other hand, even in yeast, SUMOylation of PCNA is Fomesafen site difficult to detect for the reason that only a modest proportion of PCNA is modified.