[Cell Cycle 5:6, 578-581, 16 March 2006]; ©2006 Landes Bioscience
Common Fragile Sites Nested at the Interfaces of Early and Late-Replicating Chromosome Bands Extra View
Cis Acting Components of the G2/M Checkpoint? ABSTRACT
Common fragile sites (CFS) are evolutionary conserved loci where damage appears recurrently upon treatments perturbing DNA synthesis. Although long studied, the mechanisms underlying CFS fragility are still incompletely understood and CFS function is unknown. We have mapped most of them at the junction of chromosomal bands replicating at different times in S phase, indicating that specific replication programs take place at CFS. In good agreement with this finding, we obtained results suggesting that CFS remain incompletely replicated up to late G2, even in cells that went unperturbed through S phase. The recent demonstration that the function of ATR and its downstream targets are crucial to CFS stability may thereby indicate that mitotic onset is delayed until completion of their replication. Altogether, available results now suggest that CFS constitute integral “cis” components of the G2-M checkpoint.
*Correspondence to: Michelle Debatisse; Unité Dynamique de l'Information Génétique; Bases Fondamentales et cancer; (UMR7147 IC/CNRS/UPMC); Institut Curie; 26 rue d'Ulm; 75248 Paris, France; Tel.: +33.1.42.34.66.72; Fax: +33.1.42.34.66.74; Email:
[email protected]
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Original manuscript submitted: 02/02/06 Manuscript accepted: 02/07/06
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2Département de Systématique et Evolution; UMR 5202-CNRS; Paris, France
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1Institut Curie; CNRS; Université Pierre et Marie Curie; UMR 7147; Paris, France
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Michelle Debatisse1,* Eliane El Achkar1 Bernard Dutrillaux1,2
ACKNOWLEDGEMENTS
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We thank R. Rodstein for helpful discussions and critical reading of the manuscript. M. Debatisse’ group is supported by “La Ligue Nationale Contre Le Cancer”. Eliane El Achkar was supported by a doctoral scholarship from the Lebanese National Council for Scientific Research (2001 to 2004) and from La Ligue Nationale Contre Le Cancer (2004 to 2005).
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chromosome condensation, chromosome breaks, genome instability, calyculin A, aphidicolin, cell cycle
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KEY WORDS
Fragile sites are loci where breaks, gaps and constrictions appear recurrently on metaphase chromosomes from cells grown under particular conditions. The sites are classified as rare or common, depending on their occurrence in the population. Rare fragile sites characterized at the molecular level result from mutations leading to the expansion of unstable micro- or mini-satellites. Common fragile sites (CFS), instead, are found in all individuals of a population,1 and are often conserved across species.2-5 These properties of CFS indicate that they are genuine components of the mammalian chromosomes. CFS expression is induced by stress that perturbs DNA replication such as treatments with aphidicolin (AP),6 an inhibitor of DNA polymerases alpha, delta and epsilon. It was thereby proposed that CFS are regions along chromosomes where replication elongation is unusually susceptible to perturbation. In support of this hypothesis, several studies suggest that AP treatments delay replication at CFS longer than in the rest of the genome.7-10 Sequence analysis of the CFS cloned to date revealed that they extend over megabase-long AT-rich domains, and often overlap very large genes.11 In most CFS, rearrangements and integration sites of exogenous DNA cluster in a few hyper-fragile sub-regions, the sequences of which are enriched in peaks of high flexibility and have the potential to form hairpin structures.12 As first proposed by Laird et al., even though rare and common sites do not share specific sequence motifs, their fragility may similarly rely on the accidental formation of secondary structures which could lead to fork stalling, then replisome disassembly and finally chromosome breakage.13 In agreement with this view, recent results have shown that AP treatments trigger double-strand breaks at CFS.14,15 From bacteria to higher eukaryotes, replication forks are susceptible to slowing down or stopping at genetically programmed loci. For example, in most bacteria, the arrest of replication forks at polar “ter” sites constitutes an important step in the replication termination process.16,17 In all eukaryotes, a block to fork progression occurs at the 3’ end of the rDNA transcription units, so that most forks travel through the rDNA array in the same direction as transcription does, avoiding collision between the replication and the transcription machinery.17-20 In yeast cells, regions where replication forks slow down have been searched for by global approaches,21 and their involvement in genome instability is now well established.22-25 Cha and Kleckner have shown that the fragility of yeast replication slow zones is enhanced by inactivation of Mec1 (the ATR homologue).22 Strikingly, Casper et al. have established that the frequency of breaks at FRA3B, the most active CFS of the human genome, is strongly enhanced in ATR-deficient cells.26 This was confirmed by the recent demonstration that instability at common fragile sites occurs in vivo in cells of patients bearing a heterozygous mutation for ATR (Seckel syndrome). Cells deficient for various proteins involved in the recognition and processing of stalled replication forks, such as
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BRCA1 and members of the FANC complex, also display enhanced frequencies of breaks at CFS.27 Altogether, these results suggest that most CFS might be replication slow zones or fork pause sites of the eukaryotic genomes, prone to fork collapse and breakage upon replication stress.27,28 Recently, we have been studying whether the position of CFS correlates with specific regions of the chromosomes. This work stems from earlier cytological observations on CFS. About two decades ago, Yunis et al localized recurrent breakpoints induced by various replication stress on G-banded chromosomes of human lymphocytes.6 The authors mapped 110 CFS, all of them in early replicating bands (R bands). Ten years before, B. Dutrillaux’s group had shown that breakpoints are almost systematically mapped within G bands on R-banded chromosomes, while they were localized within R bands on G-banded chromosomes.29 Hence, breaks were preferentially assigned to light bands using both types of chromosome staining (Fig. 1). This suggests that the available maps of CFS are biased by a few megabases, i.e., about the size of a prometaphase chromosome band. We thereby reassessed the localization of CFS on metaphase chromosomes of AP-treated human lymphocytes, using both R- and G-banding. Our results confirm the existence of the bias described above, and strongly suggest that the vast majority of CFS lie at the interface of R and G bands, hence at the limit of chromosomal domains replicating at different times during S phase. This conclusion was confirmed by FISH localization of probes overlapping the more active CFS of the human genome, FRA3B, FRA16D and FRAXB on prometaphase lymphocyte chromosomes.30 How replication forks behave at the junction of early and late replicating domains is still unclear. One possibility is that forks originating from the early replicating R-band stall or slow down within polar pause sites, causing sequences located downstream of these regions to be replicated later by forks coming from the coalescent G-band. As discussed above, fork progression might be blocked by accidental formation of secondary structures, as has been observed in S. cerevisiae31-33 and human cells,1,34 or by specific protein-DNA complexes, as found at genetically programmed pause sites.16-18 Alternatively, forks emanating from the early domain may enter the late domain unimpeded, which leads to passive replication and thereby inactivation of some late origins (a process called origin interference). In this case, accidental replisome disassembly may result from the long distances these forks have to cover before merging with forks from the late domain. Both the “pausing” and the “interference” mechanisms are consistent with the key role played by ATR in CFS stability.26 Noticeably, not all band interfaces are equally fragile, which suggests that different mechanisms of transition may take place at different interfaces. Moreover, not all CFS lie at the limit of R and G bands, leaving open the possibility that different mechanisms operate at different types of sites. The fact that CFS often appear as nonstaining gaps in metaphase chromosomes has long suggested that they are regions of unusual chromatin structure.35,36 In support of this hypothesis, Musio et al. have recently shown that depletion of the SMC1 cohesin leads to FRA3B instability.14 We have studied the impact of premature chromosome condensation on CFS stability. We took advantage of the fact that short treatments (30-50 min) of human lymphocytes with calyculin A (Cal), a specific inhibitor of serine-threonine phosphatases of types 1 and 2A, can trigger condensation at any stage of the cell cycle.37,38 Chromosomes of Cal-treated cells present a single chromatid with a spiral aspect when condensed in G1 phase, and resemble mitotic chromosomes when condensed in G2 phase www.landesbioscience.com
Figure 1. Ideograms of R- and G-banded human chromosome 3. Arrows point to the regions where FRA3B was mapped with each type of staining.
(Fig. 2A and B). Because they are not easily distinguishable from each other, metaphase chromosomes and chromosomes condensed in G2 have been collectively named “mitotic-like chromosomes”. We confirmed the existence of interspersed blocks of condensed and noncondensed chromatin in cells treated in S phase (Fig. 2C and D), and demonstrated that this pattern results from an inability of nonreplicated DNA to condense properly.30 Why nonreplicated DNA is incompetent for condensation in S phase, and probably also in G2, while it condenses efficiently in G1 is not known. The impact of Cal treatment on replication has been studied in different experimental models. Someya et al studied the replication of Xenopus eggs in vitro. They have shown that Cal perturbs the G1-S transition,39 but does not notably affect S phase progression when added after S phase entry.40 We confirmed the latter point by showing that DNA synthesis does not slow down in human lymphocytes challenged with Cal.30 Hence, the replication machinery proceeds normally in the presence of the drug, probably in part because nonreplicated DNA fails to condense. Besides, neither S phase completion nor G2 onset is affected by Cal treatment.30 We have observed that mitotic-like chromosomes display breaks and gaps that colocalize with AP-induced lesions, showing that the stability of CFS is compromised, whether AP delays replication or Cal advances condensation. Moreover, cells treated with Cal in the second half of G2 phase also display lesions at CFS, indicating that
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Figure 3. Cal-induced breaks in mitotic-like chromosomes. Cells were grown for 1 hour in the presence of BrdU before Cal treatment. BrdU is revealed in blue. Arrows point to some breaks. Left: only the latest bands of the late X chromosome are blue (detailed pattern is shown). Such cells were challenged with Cal in late S or early G2. Right: cell without BrdU labeling, this cell has exited S phase at least 1 hour before Cal treatment.
Figure 2. Cal-induced chromosome condensation. (A) G1, (B) G2, (C) early S phase, (D) late S phase. Staining: DNA is counterstained in blue (DAPI). Red staining: SMC2 condensin.
specific features responsible for sensitivity to condensation are present at CFS in cells that went unperturbed through S phase and early G2 (Fig. 3). The fact that breaks and gaps are infrequently observed in mitotic chromosomes of untreated cells, the chromosomes of which condense at the appropriate time, implies that these features normally disappear before mitotic onset. In agreement with this view, the later Cal is added in G2, the fewer CFS are induced.30 The molecular basis of these features is not fully elucidated. Premature chromosome condensation may simply allow observation of nonrepaired doublestrand breaks. As discussed above, the regions of transition between early and late replicating domains may be prone to fork collapse and breakage, even in the absence of replication stress. However, the demonstration that the loss of ATM function does not impact on CFS stability26 strongly argues against this hypothesis. The specific role of ATR and its downstream targets is better explained if the replication of hyper-fragile sub-regions of CFS does not take place during S phase, even in unperturbed cells. Hence, these sub-regions would remain incompetent for condensation until replication completion and subsequent chromatin reorganization that may occur up to late G2. Our results raise new questions about the relationships between such atypical timings of replication and the localization of CFS at the interface of R and G bands. For example, FRA3B overlaps the boundary of bands 3p14.2 and 3p14.1, which replicate about 2.5 and 5 hours respectively after S phase entry, in lymphocytes with an 8 hour-long S phase (El Achkar et al unpublished results). Surprisingly, replication completion at FRA3B is delayed by 3 to 5 hours compared to the replication time of band 3p14.1. This result may be accounted for by the “pausing” mechanism only if the pausing region slows down the progression of forks coming from both the early and the late domains. If the transition results from an “interference” mechanism, it remains to be explained why forks emanating from the early and the late replicating bands meet long after the origins of the late region have fired. Perhaps the interface of bands 3p14.2 and 3p14.1 coincides with a large region devoid of replication origins, or at least of origins firing during S phase. The 580
technique of molecular combing, which has been adapted to the study of large genomic regions,41 now offers the possibility to investigate further these different models. The fact that CFS expression triggers chromosome rearrangements and thereby promotes tumor progression is now largely recognized,42-45 however, little is known about their function in normal cells. Since both the sites and the very large genes nested in these loci are conserved among species, Smith et al have recently proposed that CFS and associated genes function together as a global stress response system.11 Another possibility arises from our work showing that some sub-regions of CFS might be the very last chromosomal regions to replicate,30 and from the demonstration by Glover’s team that checkpoint proteins monitoring the completion of replication are crucial to CFS stability.27 This altogether suggests that, in untreated cells, these proteins contribute to delay mitotic onset until complete duplication of CFS. We thereby propose that CFS constitute integral “cis” components of the G2-M checkpoint. Their replication would signal that replication is definitively completed and license mitotic entry. References 1. Sutherland GR, Baker E, Richards RI. Fragile sites still breaking. Trends Genet 1998; 14:501-6. 2. Glover TW, Hoge AW, Miller DE, Ascara-Wilke JE, Adam AN, Dagenais SL, Wilke CM, Dierick HA, Beer DG. The murine Fhit gene is highly similar to its human orthologue and maps to a common fragile site region. Cancer Res 1998; 58:3409-14. 3. Krummel KA, Denison SR, Calhoun E, Phillips LA, Smith DI. The common fragile site FRA16D and its associated gene WWOX are highly conserved in the mouse at Fra8E1. Genes Chromosomes Cancer 2002; 34:154-67. 4. Rozier L, El-Achkar E, Apiou F, Debatisse M. Characterization of a conserved aphidicolin-sensitive common fragile site at human 4q22 and mouse 6C1: Possible association with an inherited disease and cancer. Oncogene 2004; 23:6872-80. 5. Ruiz-Herrera A, Garcia F, Fronicke L, Ponsa M, Egozcue J, Caldes MG, Stanyon R. Conservation of aphidicolin-induced fragile sites in Papionini (Primates) species and humans. Chromosome Res 2004; 12:683-90. 6. Yunis JJ, Soreng AL, Bowe AE. Fragile sites are targets of diverse mutagens and carcinogens. Oncogene 1987; 1:59-69. 7. Le Beau. Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: Implications for the mechanism of fragile site induction. Human Molecular Genetics 1998b; 7:755-61. 8. Wang L, Darling J, Zhang JS, Huang H, Liu W, Smith DI. Allele-specific late replication and fragility of the most active common fragile site, FRA3B. Hum Mol Genet 1999; 8:431-7. 9. Hellman A, Rahat A, Scherer SW, Darvasi A, Tsui LC, Kerem B. Replication delay along FRA7H, a common fragile site on human chromosome 7, leads to chromosomal instability. Mol Cell Biol 2000; 20:4420-7. 10. Palakodeti A, Han Y, Jiang Y, Le Beau MM. The role of late/slow replication of the FRA16D in common fragile site induction. Genes Chromosomes Cancer 2004; 39:71-6.
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Mol Microbiol 1999; 31:1611-8. 17. Rothstein R, Michel B, Gangloff S. Replication fork pausing and recombination or “gimme a break”. Genes Dev 2000; 14:1-10. 18. Kobayashi T. The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork. Mol Cell Biol 2003; 23:9178-88. 19. Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 2003; 424:1078-83. 20. Calzada A, Hodgson B, Kanemaki M, Bueno A, Labib K. Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev 2005; 19:1905-19. 21. Raghuraman MK, Winzeler EA, Collingwood D, Hunt S, Wodicka L, Conway A, Lockhart DJ, Davis RW, Brewer BJ, Fangman WL. Replication dynamics of the yeast genome. Science 2001; 294:115-21. 22. Cha RS, Kleckner N. ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones. Science 2002; 297:602-6. 23. Versini G, Comet I, Wu M, Hoopes L, Schwob E, Pasero P. The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication. Embo J 2003; 22:1939-49. 24. Koszul R, Caburet S, Dujon B, Fischer G. Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments. Embo J 2004; 23:234-43. 25. Tourriere H, Versini G, Cordon-Preciado V, Alabert C, Pasero P. Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol Cell 2005; 19:699-706. 26. Casper AM, Nghiem P, Arlt MF, Glover TW. ATR regulates fragile site stability. Cell 2002; 111:779-89. 27. Glover TW, Arlt MF, Casper AM, Durkin SG. Mechanisms of common fragile site instability. Hum Mol Genet 2005; 14(Suppl 2):R197-205. 28. Cimprich KA. Fragile sites: Breaking up over a slowdown. Curr Biol 2003; 13:R231-3. 29. 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Lebofsky R, Bensimon A. DNA replication origin plasticity and perturbed fork progression in human inverted repeats. Mol Cell Biol 2005; 25:6789-97. 35. Sutherland GR. Rare fragile sites. Cytogenet Genome Res 2003; 100:77-84. 36. Wang Y. Chromatin structure of human chromosomal fragile sites. Cancer Lett 2006; 232:70-8. 37. Alsbeih G, Raaphorst GP. Differential induction of premature chromosome condensation by calyculin A in human fibroblast and tumor cell lines. Anticancer Res 1999; 19:903-8. 38. Kanda R, Eguchi-Kasai K, Hayata I. Phosphatase inhibitors and premature chromosome condensation in human peripheral lymphocytes at different cell-cycle phases. Somat Cell Mol Genet 1999; 25:1-8. 39. Someya A, Tanaka N, Okuyama A. Inhibition of initiation of DNA replication in Xenopus egg extracts by a phosphatase inhibitor, calyculin A. Biochem Biophys Res Commun 1993; 196:85-91. 40. Someya A, Tanaka N, Okuyama A. 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