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Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site

Abstract

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress1. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions2 and hotspots for chromosomal rearrangements in various cancers3. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks4. Here we show, in contrast to this view, that the fragility of FRA3B—the most active common fragile site in human lymphocytes—does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting5,6 and replication timing are highly plastic7,8 in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes1. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.

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Figure 1: Comparison of fork properties in the FHIT locus and in the bulk genome in JEFF cells.
Figure 2: Mapping of initiation and termination events along the FHIT locus.
Figure 3: Relationship between replication profile of the FHIT locus and FRA3B fragility in lymphoblastoid and fibroblastic cells.

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Acknowledgements

We thank E. Blackburn for critical reading of the manuscript. We thank Genomic Vision for making the DNA combing technology available to us. We acknowledge the Nikon Imaging Centre at Institut Curie-CNRS. We thank C. Rouzaud for help in combing experiments. A.L. is supported by a grant from the ARC (Association pour la recherche sur le cancer). The M.D. team is supported by La Ligue Nationale contre le Cancer (LNCC) (Equipe Labellisée EL2008.LNCC/MD), INCa (Institut National du Cancer) (2009-1-PLBIO-10-IC-1) and the PIC Réplication, Instabilité Chromosomique et Cancer (Institut Curie).

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Authors

Contributions

A.L. performed and analysed combing experiments. G.A.M. performed statistical and Repli-Seq analyses. S.K., A.-M.L. and O.B. performed cytogenetic analyses. N.V. and B.M. designed the Morse code. R.S.H. contributed to Repli-Seq analysis. A.L., G.A.M., O.B. and M.D. wrote the paper. M.D. planned the project.

Corresponding author

Correspondence to Michelle Debatisse.

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The authors declare no competing financial interests.

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Letessier, A., Millot, G., Koundrioukoff, S. et al. Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site. Nature 470, 120–123 (2011). https://doi.org/10.1038/nature09745

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