Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. 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 breaks. 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 setting and replication timing are highly plastic 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 lymphocytes. 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.

Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.

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Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented, but much less is known about the mechanisms controlling the spacing of initiation events(2,3), 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.

Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability.

Summary.We describe the long-term follow-up of 50 Fanconi's anaemia patients who were transplanted from a related donor with a median follow-up of >6 years. The survival estimate was 74·4% at 54 months and 58·5% at 100 months. All patients were conditioned with low-dose cyclophosphamide and thoraco-abdominal irradiation. Acute graft-versus-host disease (GvHD) of grade II or more developed in 26 patients and chronic GvHD developed in 30/43 (69·9%) patients. The survival of patients without chronic GvHD (n ¼ 13) was 100%. In addition to chronic GvHD, 20 pre-transplant transfusions was shown to have an adverse impact on survival by multivariate analysis (relative risk ¼ 7·08, P ¼ 0·0003). Prospective follow-up of growth and endocrine function could be performed in 31 patients. Of 20 boys, six have already reached normal puberty within the expected time. Among the 11 girls, three were at the pubertal age at the time of analysis. Growth retardation was common, whereas late complications (e.g. peripheral hypothyroidism, cataract) were rare. However, the most important long-term complication was the occurrence of cancer in seven patients (8-year projected incidence 24%).Among the 32 survivors, 27 (84·5%) had a normal and four a moderately reduced performance status, and all achieved complete engraftment with donor cells. Therefore transplantation was able to cure these patients who remain at high risk for developing late complications. Clearly, a genetic predisposition and chronic GvHD could have led to the development of these cancers. However, we cannot completely rule out irradiation as a cofactor in the genesis of these cancers, and therefore no longer use irradiation for the conditioning of Fanconi's anaemia patients.

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Common fragile sites have been mapped primarily in lymphocytes, but recent analyses show that the setting of these sites relies on cell type-dependent replication programs. Using a new approach, we molecularly mapped common fragile sites in human fibroblasts and showed that commitment to fragility depends on similar replication features in fibroblasts and lymphocytes, although different loci are committed in each cell type. Notably, the common fragile sites that we identified overlapped heretofore unexplained deletion clusters observed in tumors.