title: Tight Chk1 Levels Control Replication Cluster Activation in Xenopus.
abstract: DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data.
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Platel
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Arach
authors.initials: A
authors.lastname: Goldar
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Wiggins
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Pedro
authors.initials: P
authors.lastname: Barbosa
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Libeau
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Priam
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Narassimprakash
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Xenia
authors.initials: X
authors.lastname: Grodzenski
authors.affiliation: Department of Genome Biology, Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Paris South University, Gif sur Yvette, France.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Marheineke
mesh_terms: D000818:Animals; D000071877:Checkpoint Kinase 1; D003198:Computer Simulation; D004261:DNA Replication; D005260:Female; D008297:Male; D008954:Models, Biological; D010063:Ovum / Q000166:cytology / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D016196:S Phase; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*; D015854:Up-Regulation; D014981:Xenopus / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins
chemical_list: D029867:Xenopus Proteins; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus