Rif1 restrains the rate of replication origin firing in Xenopus laevis
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1.
California Institute of Technology
Abstract
Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles. Here, we find that in the absence of Rif1, patterns of replication foci change along with the acceleration of replication cluster activation. However, initiations increase only moderately inside active clusters. Our numerical simulations suggest that the absence of Rif1 compresses the temporal program towards more homogeneity and increases the availability of limiting initiation factors. We experimentally demonstrate that Rif1 depletion increases the chromatin-binding of the S phase kinase Cdc7/Drf1, the firing factors Treslin, MTBP, Cdc45, RecQL4, and the phosphorylation of both Treslin and MTBP. We show that Rif1 globally, but not locally, restrains the replication program in early embryos, possibly by inhibiting or excluding replication factors from chromatin.
Copyright and License
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Acknowledgement
We thank J. Walter for the gift of XCdc7 antibody. Anti-Xenopus Orc2 antibody, developed by JL. Maller was obtained from the European Xenopus Resource Centre, curated with funding from the Wellcome Trust/BBSRC and maintained by the University of Portsmouth, School of Biological Sciences. We are grateful to A. Donval for her help with frog fertilizations. We thank B. Miroux and J.-L. Ferrat for critical reading of the paper. This research was supported by grants to OB from ARC (Association pour la Recherche sur le Cancer), Association Retina France, Fondation Valentin Haüy, and UNADEV (Union Nationale des Aveugles et Déficients Visuels) in partnership with ITMO NNP (Institut Thématique Multi-Organisme Neurosciences, sciences cognitives, neurologie, psychiatrie)/AVIESAN (alliance nationale pour les sciences de la vie et de la santé). D.C. had an IDEX Paris-Saclay University Ph.D. fellowship. Work in the laboratory of W.G.D. is supported by National Institutes of Health grant R01 GM043974.
Contributions
O.H. designed, performed, supervised and analyzed experiments and contributed to the writing of the paper, D.C. analyzed experiments and designed the model and the computational framework, H.N. performed experiments, O.B. designed and performed experiments, A.K. and W.G.D. provided critical reagents, A.G. designed the model and the computational framework and supervised the analysis, K.M. designed, performed, analyzed, supervised experiments and wrote the paper. All authors discussed the results and contributed to the final paper.
Data Availability
All data are included in this paper and its supplementary information files. Numerical source data for all graphs and charts are provided in the Supplementary Data file. Uncropped Western blot images are presented in Supplementary Fig. 8. Other information about this study are available from the corresponding author upon reasonable request.
Code Availability
The code of simulation and multivariable fitting used in this study, initially published in41, is deposited on GitHub (https://github.com/DidiCi/MMsimulation).
Conflict of Interest
The authors declare no competing interests.
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Additional details
- PMCID
- PMC10387115
- Wellcome Trust
- Biotechnology and Biological Sciences Research Council
- Retina France
- Université Paris-Saclay
- National Institutes of Health
- R01 GM043974
- Caltech groups
- Division of Biology and Biological Engineering