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Replication fork movement sets chromatin loop size and origin choice in mammalian cells


Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented1, but much less is known about the mechanisms controlling the spacing of initiation events2,3, namely the completion of DNA replication. Here we show that origin use in Chinese hamster cells depends on both the movement of the replication forks and the organization of chromatin loops. We found that slowing the replication speed triggers the recruitment of latent origins within minutes, allowing the completion of S phase in a timely fashion. When slowly replicating cells are shifted to conditions of fast fork progression, although the decrease in the overall number of active origins occurs within 2 h, the cells still have to go through a complete cell cycle before the efficiency specific to each origin is restored. We observed a strict correlation between replication speed during a given S phase and the size of chromatin loops in the next G1 phase. Furthermore, we found that origins located at or near sites of anchorage of chromatin loops in G1 are activated preferentially in the following S phase. These data suggest a mechanism of origin programming in which replication speed determines the spacing of anchorage regions of chromatin loops, that, in turn, controls the choice of initiation sites.

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Figure 1: The density of active origins depends on replication speed.
Figure 2: oriGNAI3 prominence appears only during the second S phase after shifting 474 cells to fast conditions.
Figure 3: Large halos and preferential binding of oriGNAI3 to the nuclear matrix in G1 of ‘fast’ 474 cells.

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We thank E. Blackburn, R. Rothstein and F. Toledo for discussions and critical reading of the manuscript, and Genomic Vision for making available the DNA combing technology. S.C. is supported by a grant from the ARC (Association pour la Recherche sur le Cancer), and S.G. and N.A. are supported by a grant from the Ministère de la Recherche. The M.D. team is supported by La Ligue Nationale contre le Cancer and the Agence Nationale de la Recherche (ANR).

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Correspondence to Michelle Debatisse.

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This file contains Supplementary Figures 1-8 with Legends. The Supplementary Figure 1 includes a model for the control of origin usage in mammalian somatic cells. (PDF 6942 kb)

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Courbet, S., Gay, S., Arnoult, N. et al. Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature 455, 557–560 (2008).

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