8
Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site.
Nature. 2011 Feb 3; 470(7332):120-3
All selected articles haveThis article has been added to your saved articles
You can follow/unfollow articles via MyF1000 or via the article or browse pages.
21 Feb 2011
DOI: 10.3410/f.8492962.8964063
In a technical tour de force, Debatisse and colleagues provide the first direct experimental evidence that common fragile sites (CFSs) are prone to breakage because of their unusual replication profile.
CFSs are large chromosomal regions that frequently break under replication stress. Despite the fact...Continue reading
CFSs are large chromosomal regions that frequently break under replication stress. Despite the fact...Continue reading
In a technical tour de force, Debatisse and colleagues provide the first direct experimental evidence that common fragile sites (CFSs) are prone to breakage because of their unusual replication profile.
CFSs are large chromosomal regions that frequently break under replication stress. Despite the fact that CFS fragility plays an active role in the cancer process, the molecular mechanisms involved have remained largely unexplored.
In this manuscript, Debatisse and colleagues have combined single-molecule analysis and microarray-based assays to monitor the replication of FRA3B, one of the best-characterized human CFSs. Using DNA combing, they have mapped individual replication forks, active origins and termination sites within a 1.6Mb region spanning the most fragile domain of FRA3B. This analysis revealed that replication forks do not pause at this locus, arguing against the existence of ‘replication slow zones’ blocking elongation. In contrast, the authors show that the most fragile region of FRA3B is passively replicated from very late origins located outside of this 670Kb core domain. Interestingly, this unusual replication pattern is only detected in lymphoblastoid cells, but not in fibroblasts, in which FRA3B is actively replicated from internal origins and does not break.
Together, these data indicate that CFSs correspond to large regions devoid of replication origins that replicate very late in S phase. Since origin usage varies with cell type, this work also explains the tissue specificity of CFS instability.
CFSs are large chromosomal regions that frequently break under replication stress. Despite the fact that CFS fragility plays an active role in the cancer process, the molecular mechanisms involved have remained largely unexplored.
In this manuscript, Debatisse and colleagues have combined single-molecule analysis and microarray-based assays to monitor the replication of FRA3B, one of the best-characterized human CFSs. Using DNA combing, they have mapped individual replication forks, active origins and termination sites within a 1.6Mb region spanning the most fragile domain of FRA3B. This analysis revealed that replication forks do not pause at this locus, arguing against the existence of ‘replication slow zones’ blocking elongation. In contrast, the authors show that the most fragile region of FRA3B is passively replicated from very late origins located outside of this 670Kb core domain. Interestingly, this unusual replication pattern is only detected in lymphoblastoid cells, but not in fibroblasts, in which FRA3B is actively replicated from internal origins and does not break.
Together, these data indicate that CFSs correspond to large regions devoid of replication origins that replicate very late in S phase. Since origin usage varies with cell type, this work also explains the tissue specificity of CFS instability.
Disclosures
None declared
Add Comment
Pasero P and Lengronne A: F1000Prime Recommendation of [Letessier A et al., Nature 2011, 470(7332):120-3]. In F1000Prime, 21 Feb 2011; DOI: 10.3410/f.8492962.8964063. F1000Prime.com/8492962#eval8964063
18 Jul 2011
Olivier Hyrien
F1000 Cell Biology
Institut de Biologie de l'ENS, CNRS UMR8197, Inserm U1024,
Paris, France.
F1000 Cell Biology
Institut de Biologie de l'ENS, CNRS UMR8197, Inserm U1024,
Paris, France.
Controversial, New Finding
DOI: 10.3410/f.8492962.13026055
This study shows that the chromosomal FRA3B locus is much more fragile in lymphocytes than in fibroblasts and replicates differently in both cell types. Chromosomal fragility is proposed to result from the combination of a late replication timing and a lack of replication initiation events over a 700kb...Continue reading
This study shows that the chromosomal FRA3B locus is much more fragile in lymphocytes than in fibroblasts and replicates differently in both cell types. Chromosomal fragility is proposed to result from the combination of a late replication timing and a lack of replication initiation events over a 700kb region in lymphocytes. Common fragile sites (CFSs) are loci present in all individuals that preferentially exhibit gaps and breaks on metaphase chromosomes following partial inhibition of DNA synthesis {1}. CFSs are sites of frequent chromosome breakage and rearrangement in cancers.
Late replication is a characteristic feature of CFSs, but many other regions of the genome that also replicate late do not exhibit fragility. Several CFSs lie within large genes and CFSs seem particularly sensitive to further replication delay in response to replication stress. It has been proposed that the formation of secondary structures that inhibit replication fork progression, or replication-transcription collisions, or a particular organization of replication origins may form the molecular basis for fragility.
Letessier et al. used DNA combing to examine the replication dynamics of 1.6Mb of DNA spanning FRA3B, the most fragile CFS, in human lymphocytes. They found that replication fork speeds are heterogeneous along the locus, but not any more than over the bulk genome, and that slow or stalled forks do not cluster in any particular subregion in either untreated or aphidicolin-treated cells (aphidicolin is a DNA polymerase inhibitor routinely used to reveal fragile sites). In contrast, initiation events are not evenly spread over the locus. No initiation event is found within a 700kb region termed the FRA3B core, whereas multiple initiations are observed on both sides of this core. Forks travel in both directions within the core and termination events are evenly distributed along the entire locus. Slowing replication with aphidicolin triggers the recruitment of latent origins in the flanking regions, but not in the core, to a level comparable to the bulk genome. Thus, fragility appears to be associated with a lack of replication origins over a large region.
The analysis was pushed further by exploiting recent replication timing data obtained by sequencing newly replicated DNA in both lymphocytes and fibroblasts. Although the locus replicates late in both cell types, the replication timing profiles suggested that initiation events occur over the FRA3B core in fibroblasts. Letessier et al. turned back to DNA combing to demonstrate that indeed, in both untreated and aphidicolin-treated fibroblasts, initiation and termination events are evenly distributed along the locus. They then used molecular cytogenetics to demonstrate that the frequency of breaks at the FRA3B locus after aphidicolin treatment is at least ten times lower in fibroblasts than in lymphocytes. Similar results are found for FRA16D, the second most active CFS in lymphocytes. It is concluded that fragility requires the combination of a late replication timing and a lack of initiation events over a large region.
Interestingly, an independent DNA combing study of the FRA16C CFS by Ozeri-Galai et al. concluded that the fragility of this region is not due to an inherent lack of initiation events but to forks stalling at AT-rich regions and to an inability to activate additional origins upon replication stress {2}. The common hallmark of CFSs may, therefore, not be an inherent lack of origins but a failure to activate enough rescuing origins when replication is slowed.
Late replication is a characteristic feature of CFSs, but many other regions of the genome that also replicate late do not exhibit fragility. Several CFSs lie within large genes and CFSs seem particularly sensitive to further replication delay in response to replication stress. It has been proposed that the formation of secondary structures that inhibit replication fork progression, or replication-transcription collisions, or a particular organization of replication origins may form the molecular basis for fragility.
Letessier et al. used DNA combing to examine the replication dynamics of 1.6Mb of DNA spanning FRA3B, the most fragile CFS, in human lymphocytes. They found that replication fork speeds are heterogeneous along the locus, but not any more than over the bulk genome, and that slow or stalled forks do not cluster in any particular subregion in either untreated or aphidicolin-treated cells (aphidicolin is a DNA polymerase inhibitor routinely used to reveal fragile sites). In contrast, initiation events are not evenly spread over the locus. No initiation event is found within a 700kb region termed the FRA3B core, whereas multiple initiations are observed on both sides of this core. Forks travel in both directions within the core and termination events are evenly distributed along the entire locus. Slowing replication with aphidicolin triggers the recruitment of latent origins in the flanking regions, but not in the core, to a level comparable to the bulk genome. Thus, fragility appears to be associated with a lack of replication origins over a large region.
The analysis was pushed further by exploiting recent replication timing data obtained by sequencing newly replicated DNA in both lymphocytes and fibroblasts. Although the locus replicates late in both cell types, the replication timing profiles suggested that initiation events occur over the FRA3B core in fibroblasts. Letessier et al. turned back to DNA combing to demonstrate that indeed, in both untreated and aphidicolin-treated fibroblasts, initiation and termination events are evenly distributed along the locus. They then used molecular cytogenetics to demonstrate that the frequency of breaks at the FRA3B locus after aphidicolin treatment is at least ten times lower in fibroblasts than in lymphocytes. Similar results are found for FRA16D, the second most active CFS in lymphocytes. It is concluded that fragility requires the combination of a late replication timing and a lack of initiation events over a large region.
Interestingly, an independent DNA combing study of the FRA16C CFS by Ozeri-Galai et al. concluded that the fragility of this region is not due to an inherent lack of initiation events but to forks stalling at AT-rich regions and to an inability to activate additional origins upon replication stress {2}. The common hallmark of CFSs may, therefore, not be an inherent lack of origins but a failure to activate enough rescuing origins when replication is slowed.
References
Disclosures
None declared
Hyrien O: F1000Prime Recommendation of [Letessier A et al., Nature 2011, 470(7332):120-3]. In F1000Prime, 18 Jul 2011; DOI: 10.3410/f.8492962.13026055. F1000Prime.com/8492962#eval13026055
14 Sep 2011
New Finding, Refutation
DOI: 10.3410/f.8492962.13640055
Common fragile sites are large chromosomal regions that are prone to breakage under conditions of replication stress. Although they have been implicated in chromosome rearrangements in tumours {1} and shown to be preferential targets of allelic imbalance in preneoplastic lesions {2,3}, the underlying...Continue reading
Common fragile sites are large chromosomal regions that are prone to breakage under conditions of replication stress. Although they have been implicated in chromosome rearrangements in tumours {1} and shown to be preferential targets of allelic imbalance in preneoplastic lesions {2,3}, the underlying mechanisms by which they contribute to tumorigenesis have not been well defined. Using elegant methods, the authors show that it is the scarcity of replication initiation that contributes to breaks occuring at fragile sites and that this phenomenon is cell-type-specific.
Common fragile sites have been implicated in tumorigenesis and a variety of reasons have been put forward for the breaks observed at common fragile sites. For instance, it has been suggested that the sequence at common fragile sites may lead to secondary structure formation and pose as potential replication fork barriers that may cause fork slowing/stalling and eventual fork collapse, leading to breaks at these sites {1}.
However, this study points to a different reason and conclusively shows by using FRA3B, the most common fragile site in human lymphocytes, that it is the paucity of replication initiation events that is instead responsible. The authors use DNA combing in combination with FISH to map active origins and termination sites within the FHIT locus, which overlaps with the FRA3B common fragile site, as well as for determination of replication fork speed and potential fork stalling along the locus.
Their studies revealed that forks do not stall at this locus, arguing against stalled replication forks as the cause of damage at the common fragile sites. Instead, they observe a deficit in replication origin density within the FRA3B core, with initiation events occurring only at the regions flanking the core. Indeed, further studies using fibroblasts revealed that replication initiation events do occur normally within the FRA3B locus in fibroblasts and, consistent with this, breaks are not detected at this locus.
In all, this study is exciting as it offers a plausible molecular mechanism for breaks seen at common fragile sites. Thus, differences in replication origin usage and replication initiation in different cell types may represent the underlying reason for differential fragility seen in different cell types. It remains to be seen whether the lack of completely replicated fragile sites could contribute to genomic instability and cancers via induction of DNA breaks arising, for example, by premature chromosome condensation {4} at these sites.
Common fragile sites have been implicated in tumorigenesis and a variety of reasons have been put forward for the breaks observed at common fragile sites. For instance, it has been suggested that the sequence at common fragile sites may lead to secondary structure formation and pose as potential replication fork barriers that may cause fork slowing/stalling and eventual fork collapse, leading to breaks at these sites {1}.
However, this study points to a different reason and conclusively shows by using FRA3B, the most common fragile site in human lymphocytes, that it is the paucity of replication initiation events that is instead responsible. The authors use DNA combing in combination with FISH to map active origins and termination sites within the FHIT locus, which overlaps with the FRA3B common fragile site, as well as for determination of replication fork speed and potential fork stalling along the locus.
Their studies revealed that forks do not stall at this locus, arguing against stalled replication forks as the cause of damage at the common fragile sites. Instead, they observe a deficit in replication origin density within the FRA3B core, with initiation events occurring only at the regions flanking the core. Indeed, further studies using fibroblasts revealed that replication initiation events do occur normally within the FRA3B locus in fibroblasts and, consistent with this, breaks are not detected at this locus.
In all, this study is exciting as it offers a plausible molecular mechanism for breaks seen at common fragile sites. Thus, differences in replication origin usage and replication initiation in different cell types may represent the underlying reason for differential fragility seen in different cell types. It remains to be seen whether the lack of completely replicated fragile sites could contribute to genomic instability and cancers via induction of DNA breaks arising, for example, by premature chromosome condensation {4} at these sites.
References
1.
Chromosome fragile sites.
Annu Rev Genet 2007; (41):169-92
PMID: 17608616
2.
Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.
Nature 2005 Apr 14; 7035(434):907-13
PMID: 15829965
3.
DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.
Nature 2005 Apr 14; 7035(434):864-70
PMID: 15829956
4.
Premature condensation induces breaks at the interface of early and late replicating chromosome bands bearing common fragile sites.
Proc Natl Acad Sci USA 2005 Dec 13; 50(102):18069-74
PMID: 16330769
Disclosures
None declared
Pellman D and Crasta K: F1000Prime Recommendation of [Letessier A et al., Nature 2011, 470(7332):120-3]. In F1000Prime, 14 Sep 2011; DOI: 10.3410/f.8492962.13640055. F1000Prime.com/8492962#eval13640055
F1000Prime Recommendations, Dissents and Comments for [Letessier A et al., Nature 2011, 470(7332):120-3]. In F1000Prime, 26 May 2013; F1000Prime.com/8492962
Only registered/signed-in users can make comments.
Sign in or Subscribe to create your user account and join the discussion.
Sign in or Subscribe to create your user account and join the discussion.
No comments yet.
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.
DOI: 10.1038/nature09745
PMID: 21258320
