The FRA3B at 3p14.2 is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin or folate stress. The molecular basis for chromosome fragility at FRA3B is unknown. In contrast to the rare fragile sites, including FRAXA, no repeat motifs, such as trinucleotide repeats, have been identified within FRA3B. Several lines of evidence suggest that fragile sites are regions of DNA whose replication is unusually sensitive to interference. We have used fluorescence in situ hybridization to determine the relative timing of replication of FRA3B sequences. Our studies revealed that FRA3B sequences are late replicating. Exposure to aphidicolin, an inhibitor of both DNA polymerase [alpha] and [delta], results in a reproducible delay in the timing of replication, and some cells enter G2 without having completed replication of FRA3B sequences. Our results support a model in which common fragile sites are sequences that initiate replication late in S phase or are slow to replicate, and the chromosomal breaks and gaps observed in metaphase cells are due to unreplicated DNA.
Chromosomal fragile sites are specific loci that show gaps, breaks or rearrangements in metaphase chromosomes when cells are cultured under conditions that inhibit DNA replication (low folate or thymidine levels, or the presence of chemicals such as aphidicolin) (1). Fragile sites, particularly common fragile sites, exhibit several features characteristic of highly unstable or recombinogenic regions of the genome. Fragile site induction in cultured cells results in increased inter- and intrachromosomal recombination events, including increased sister chromatid exchange (2,3) and chromosomal rearrangements, such as translocations and deletions (4-6). It has been hypothesized that the common fragile sites may play a mechanistic role in the chromosomal abnormalities and genetic mutations observed in human tumor cells (7).
Until recently, essentially nothing was known about the DNA sequences at fragile sites. Six rare fragile sites (FRAXA, FRAXE, FRAXF, FRA16A, FRA16B and FRA11B) have been cloned and characterized (reviewed in ref. 8). With the exception of FRA16B, the mutation leading to the expression of fragility is the expansion and methylation of a CGG trinucleotide repeat. FRA16B also involves the expansion of a repeat, namely, a 33 bp AT-rich minisatellite repeat (9). In the case of the FRAXA mental retardation syndrome, trinucleotide repeat expansion is associated with chromosome fragility, abnormal methylation within the CpG island adjoining the region and absence of expression of the FMR1 gene (10). In contrast, repeat expansion in FRA11B may mediate chromosomal deletions as is suggested by the del(11q) in Jacobsen syndrome (11).
Laird et al. hypothesized that rare fragile sites are late replicating (12), and this subsequently was demonstrated for FRAXA and FRAXE, the only fragile sites for which the timing of replication has been characterized (13-15). Replication is delayed further in FRAXA and FRAXE alleles with trinucleotide repeat expansion (13-15). The changes that give rise to fragile sites on metaphase chromosomes are likely to involve the formation of complex intramolecular structures at CGG repeats, such as tetraplexes (a folded hairpin), that block DNA synthesis (16).
Although less is known about the DNA structure at common fragile sites, they have also been implicated recently in disease processes. FRA3B at 3p14.2 is the most common of the constitutive fragile sites observed when DNA replication is perturbed by aphidicolin or folate stress (17). Using independent approaches, four groups of investigators have isolated DNA sequences from FRA3B; in each case, the sequences are contained within the 850A6 yeast artificial chromosome (YAC) (Fig. 1) (18-22). Wilke et al. used a fluorescence in situ hybridization (FISH)-based approach to localize FRA3B to a region spanning >100 kb that is ~160 kb telomeric of the breakpoint in the t(3;8) identified in a family with hereditary renal carcinoma (18). Paradee et al. mapped aphidicolin-induced breakpoints in chromosome 3-containing somatic cell hybrids, and identified two clusters of breaks flanking thoseidentified by Wilke et al.(19,20). Rassool et al.used a direct cloning approach basedon the integration of transfected pSV2neo DNA into the FRA3B region to isolate ~100 kb of genomically unstable sequences ~350 kb telomeric of the t(3;8) breakpoint (21). Finally, Ohta et al. cloned the candidate tumor suppressor gene, FHIT, from the same region, and it is now known that FHIT spans FRA3B (22).
Using this FISH-based approach, mitogen-stimulated lymphocytes are pulse-labeled with bromodeoxyuridine (BrdU) followed by FISH of labeled test or control probes. S-phase nuclei are identified using BrdU-specific antibodies. Nuclei in which the DNA sequences encompassed by the probe have not yet replicated show two single hybridization signals (Fig. 2A), whereas those in which the sequences have replicated have two sets of doublets (Fig. 2C). Thus, in an asynchronous population of S-phase cells, the higher the percentage of doublet nuclei, the earlier the sequences replicate in S phase. In general, most loci replicate in a synchronous manner, with only ~10-20% of nuclei showing an asynchronous single-double pattern (Fig. 2B) (26).
FISH analysis of clones derived from three genomic contigs spanning ~350 kb revealed that FRA3B DNA sequences are late replicating. The percentage of S-phase cells with doublets ranged from 10 to 36% (mean 21.7 ± 6.4%) (Fig. 3). In contrast, an early replicating control probe had 80% doublet signals (IGHM), middle-S replicating sequences had 46-56% doublets (COS6, ABL and BCL6), and a late replicating probe had 21% doublets (FMR1) (Fig. 3). The INS probe was at the boundary of middle-S and late-S replicating (25-33% doublets).
To examine the effects of aphidicolin on replication of FRA3B sequences, we determined the timing of replication in cells exposed to aphidicolin (0.4 µM), and those without aphidicolin treatment. Aphidicolin resulted in a small, but significant, delay in the timing of replication as evidenced by a mean decrease of 4.4 percentage points (decrease from mean of 21.7 to 17.3%) in doublet signals for FRA3B probes [the ratio of the odds of both alleles having replicated with aphidicolin to those without is 0.76, 95% confidence interval (CI) 0.63-0.91]. A test of the null hypothesis that the odds ratio is equal to 1.0 yielded a P value of 0.002. An exception among FRA3B probes was HP2A, in which four replicate experiments showed no change in two analyses and small increases in two other analyses (increases of 2 and 3 percentage points).
The effect of aphidicolin on the timing of replication of the control probes was variable, with an overall increase of 2.1 percentage points in doublet signals (increase from 47.3 to 49.4%, odds ratio 1.09, 95% CI 0.84-1.41); this change was not significant (P = 0.528). For FRA3B probes, aphidicolin was estimated to decrease the odds of having replicated by 24% whereas, for control probes, it was estimated to increase the odds by 9%. The difference between the effect of aphidicolin on the control and FRA3B probes was statistically significant (P = 0.025).
To test the hypothesis that chromosome breakage results from failure to complete replication of FRA3B sequences prior to entry of the cell into G2, we evaluated the replication status of FRA3B sequences in G2 cells. Cyclin B1 levels oscillate during the cell cycle. Protein levels begin to increase in late S-early G2, and reach peak levels in late G2. The protein is degraded in late mitosis and the cell cycle is reset (29). Thus, we identified G2 cells by the detection of cyclin B1 using specific antibodies (Fig. 2D). In asynchronous cultures of mitogen-stimulated lymphocytes, 8.4% of cells were cyclin B1-positive (42/500 cells). A similar frequency was noted in lymphocytes cultured in the presence of aphidicolin (7.8%, 39/500 cells). The duration of the cell cycle in phytohemagglutinin (PHA)-stimulated lymphocytes is 12 h; G1 is ~ 4 h, S phase is 5-5.5 h, G2 is 2-3 h and mitosis is 0.5 h (30,31). Thus, G2 cells should represent between 16 and 25% of the population. The detection of cyclin B1 in ~8% of cells suggests that this assay detects only G2 cells, and may detect those in late G2 when cyclin B1 levels are the highest, rather than those in late S-early G2 or early G2.
The percentage of cyclin B1-positive cells with doublet signals ranged from 58 to 88% for FRA3B probes, and from 47 to 85% for control probes (Fig. 4). Despite the fact that these cells have completed S phase, we anticipated that this value would be substantially less than 100% due to the nature of FISH (see above, Replication analysis using FISH). In cells exposed to aphidicolin, there was a significant decrease in the percentage of cells with doublets for FRA3B probes, with the exception of [lambda]HP2A (8-29% decrease, mean decrease of 16.5 percentage points from 72.1 to 55.6%). The odds ratio is 0.48 (95% CI 0.39-0.60, P < 0.001). In contrast, the drug increased the doublet signals among the control probes by an average of 3.4 percentage points (from 68.7 to 72.1%, odds ratio 1.16, 95% CI 0.99-1.34); this change was not significant (P = 0.062) (Fig. 4). Aphidicolin was estimated to decrease the odds of having replicated by 52% for FRA3B probes, while increasing the odds by 16% for control probes. A test of the null hypothesis that the effect of aphidicolin is the same for FRA3B and control probes yields a P value of <0.001, providing strong evidence that the drug affects the FRA3B region differently from other regions of the genome examined. The results with HP2A were unusual in that aphidicolin had little effect in two experiments (2% decrease, 5% increase), but resulted in a substantial decrease (20 and 25%) in two other experiments.
We have demonstrated that the FRA3B region replicates late in S phase, and aphidicolin treatment delays replication further, resulting in failure to complete DNA replication. Mammalian cell replicons typically range in size from 50 to 330 kb (average ~100 kb) (33); thus, the FRA3B region we examined (~350 kb) may be composed of multiple replicons, within a single replication zone. Therefore, the stalling of a single replication fork, for example at an unusual DNA sequence that is difficult to replicate, is unlikely to account for the observed pattern of delayed replication. Although our analysis of the FRA3B replication pattern in S-phase cells reveals that aphidicolin induces a delay in replication, it does not determine whether there is stalling (an irreversible stop) or simply a slowing of the replication fork in a region that ultimately completes replication. However, the results of our analysis of FRA3B sequences in G2 cells imply that there is stalling of replication, and that these sequences fail to complete replication in some cells.
The nature of the inducing agents for both the rare folate-sensitive and common fragile sites suggested that the process was likely to operate during DNA synthesis (1,17); however, until now there has been little experimental documentation to support such a mechanism. A number of models for chromosome breakage at common fragile sites involving DNA replication can be considered. (i) Timing of replication: we have demonstrated that FRA3B sequences replicate late in the S phase; thus, our data support this model. (ii) Widely separated origins: at high concentrations, aphidicolin is known to cause stalling of replication forks; the distance between replication forks within the FRA3B region may be too large for unreplicated DNA to be repaired by other mechanisms. (iii) Unusual DNA sequences: the FRA3B region may be rich in DNA sequences that are difficult to replicate or that facilitate DNA breakage, such as palindromic sequences which form hairpin loops. (iv) Chromatin structure: the FRA3B region may be within tightly packed chromatin, which is difficult to replicate, or within a very large chromatin loop, and accessible to DNA damage. (v) High level of gene transcription: the FHIT gene may be transcribed at high levels in S phase, which may interfere with the activity of origins of replication or of replication fork progression. (vi) A combination of factors: the above models are not mutually exclusive.
Our results do not preclude the co-existence of other mechanisms that may facilitate breakage. The fact that aphidicolin preferentially delays replication in the FRA3B region, but not other late replicating sequences we examined, indicates that late replication alone does not account for the entire effect. Late replication in itself is likely to synergize with other phenomena, i.e. widely separated origins of replication and/or unusual DNA sequences that are difficult to replicate may compromise DNA replication within normally late replicating regions. Although there may be mechanisms normally used by the cell to overcome stalling of replication forks, e.g. replication completed by neighboring origins or the induction of secondary origins of replication, our results suggest that these mechanisms are ineffective within FRA3B sequences. It is possible that stalled replication forks are converted into double-stranded breaks within FRA3B, which may lead to aberrant recombination repair and resultant genetic instability.
Our analysis of the replication pattern of FRA3B sequences in G2 cells suggests that exposure to aphidicolin results in an increase in the percentage of cells with unreplicated FRA3B sequences, as assessed by a decrease in the proportion of cells with doublet signals. It is notable that the decrease in percentage of cells with doublets in G2 (mean 16.5%) is similar to the percentage of cells in metaphase cells expressing FRA3B in healthy individuals (mean 19%) (refs 3,17 and Le Beau et al., unpublished analysis of 32 individuals). Eukaryotic cells possess a checkpoint mechanism whereby individual cells register whether S phase has been completed before entering mitosis. Inhibition of DNA synthesis or incomplete DNA synthesis causes the cell to arrest in interphase, rather than to progress to mitosis. However, deficiency of many of the different proteins required for DNA synthesis can lead to inappropriate entry into mitosis (reviewed in ref. 34). In Schizosaccharomyces pombe, DNA polymerase [alpha] is required for a checkpoint signal that prevents mitosis until S phase has been completed. Deletion of the pol1+ gene encoding DNA polymerase [alpha] allows entry into mitosis in cells with incomplete DNA synthesis. A survey of other replication mutants suggests that replication initiation complexes, including DNA polymerase [alpha], rather than proteins involved in elongation, are the key components necessary for the checkpoint signal that delays mitosis (35).
The observation of fragile sites in metaphase cells induced by agents such as aphidicolin suggests that cells with DNA lesions have escaped the G2-M checkpoint. In some cases, the number of DNA breaks/gaps can be very numerous (>30/cell). It is not clear whether the DNA lesions are too few for recognition, or whether the types of lesions induced by aphidicolin are not recognized by the G2-M checkpoint mechanism. The checkpoint mechanism may detect certain lesions, such as double-stranded breaks, more efficiently than it detects stalled replication forks. Similarly, whether inhibition of DNA polymerase [alpha]/[delta] alters the G2-M checkpoint is not known.
In summary, our studies provide initial evidence that alteration in DNA replication is involved in the expression of aphidicolin-inducible fragile sites; however, a number of issues remain unresolved. The results of our studies suggest that inhibition of DNA polymerase [alpha] and [delta] may induce fragile sites by delaying DNA replication further in regions that are already late replicating or slow to replicate, and that failure to complete replication of FRA3B sequences before condensation of chromatin into metaphase chromosomes may give rise to the chromosomal breaks and gaps characteristic of fragile sites.
Peripheral blood samples were obtained from healthy individuals, and lymphocytes were isolated by Ficoll gradient separation. Mitogen-stimulated lymphocytes (5*106) were cultured in 10 ml of RPMI 1640, 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES and 10 µg/ml PHA (Murex Diagnostics, Inc., Dartford, UK) for 72 h at 37°C in 5% CO2/95% air. Some cultures were exposed to aphidicolin (0.4 µM; Sigma, St Louis, MO) for 25 h prior to harvesting. S-phase cells were identified by labeling with BrdU (10-5 M; Sigma) for 30-60 min prior to harvesting.
FISH was performed as described previously (36). Labeled probes were prepared by nick translation using Bio-16-dUTP or digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN), or directly labeled nucleotides (Vysis, Inc., Downers Grove, IL). Biotin-labeled probes were detected with fluoresceinisothiocyanate (FITC)-conjugated avidin, and digoxigenin-labeled probes were detected with rhodamine-conjugated sheep digoxigenin-specific antibodies (Vector Laboratories, Burlingame, CA). After the hybridization, washing and blocking steps, the slides were incubated with BrdU-specific antibodies (Becton-Dickinson, Franklin Lakes, NJ) at 37°C for 30 min, followed by washes and incubation with FITC-conjugated goat anti-mouse IgG (Jackson Immunochemicals, West Grove, PA). Cells in G2 were identified with indirect immunofluorescence using mouse monoclonal cyclin B1-specific IgG antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Cyclin B1-specific antibody was detected with biotinylated anti-mouse IgG horse antibodies (Vector Laboratories), followed by FITC-conjugated avidin. Interphase nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The slides were examined using a DAPI/FITC/rhodamine triple band pass filter (ChromaTechnology, Brattleboro, VT); 100 nuclei were scored for the presence of two singlet signals, one single and one double signal, or two signal doublets by each of two observers in a blinded fashion. Hybridizations were repeated multiple times for many of the probes.
The FRA3B clones are bacteriophage [lambda] or cosmid clones derived from genomic contigs of the FRA3B region (Fig. 1); all of the clones are contained within YAC850A6. Cosmids 1A6 and 3C3 contain two clusters of aphidicolin-induced breakpoints, and are ~100 and ~300 kb telomeric of the t(3;8) breakpoint, respectively (19,20). Clones [lambda]263, [lambda]247 and [lambda]106 contain a human papilloma virus 16 integration site, and are located between the two clusters of aphidicolin-induced breakpoints, ~160 kb telomeric to the t(3;8) breakpoint (18). Clones [lambda]4005, [lambda]5002, [lambda]HP2A and [lambda]3016 are the most telomeric clones analyzed, and are from an ~100 kb contig ~350 kb telomeric of the translocation breakpoint (21). Fragile site expression within each of these clones has been demonstrated by the analysis of chromosome breakage in cells exposed to aphidicolin (18-21). The following cosmid and [lambda] clones were kindly provided by the investigators indicated, and were used as control probes: [lambda]IGHM-H24 (IGHM, ATCC, early replicating) (26), cosmid COS6 (middle-S replicating, Michael Lerman), cosmid pCV105 (ABL, middle-S replicating, Carol Westbrook), [lambda]BCL6-C, -E and -G (BCL6, middle-S replicating, Beverly Baron), [lambda]INS-2, -3 and -5 (INS, middle-late S replicating, Graeme Bell), and cosmid C22-3 (FMR1, late replicating, Stephen Warren). The BCL6 clones span ~45 kb, and the INS clones are overlapping and span ~50 kb.
Analysis was performed on the proportion (p) of cells with doublet signals for each probe. Since each slide was read by both observers, the results were averaged to yield a single percentage estimate for each slide. A model was fit to the data in which the logarithm of the odds (p/1 - p) of both alleles having replicated was represented as a sum of terms due to aphidicolin or probe type (FRA3B versus control), and the interaction of the two. Exponentiating the coefficient for aphidicolin provides an estimate of the multiplicative change in odds due to the drug among control probes, while exponentiating the sum of the coefficients for drug and the interaction term yields the multiplicative change in odds among FRA3B probes. A Wald test of the interaction term was used to test the null hypothesis that the effect of the drug was the same for both types of probes. The variability in the observed proportion of doublets was assumed to be proportional to the binomial variance [p * (1 - p)], and the correlation within each probe was assumed constant. The model was fit using the Generalized Estimating Equations approach of Liang and Zeger (37), as implemented in the computing package Stata 5.0 (StataCorp., 1997).
We thank Elizabeth M. Davis, Anthony A. Fernald, Steven Minaglia and Cynthia Klestinec for expert technical assistance, and L. Philip Schumm for statistical analysis. This work was supported by PHS grants (CA41644, M.M.L. and T.W.M.; CA43222, T.W.G.; and CA48031, D.I.S.).
Human Molecular Genetics
Pages
Introduction
Results
Replication analysis using FISH
FRA3B sequences are late replicating
FRA3B sequences may be unreplicated in G2 cells
Discussion
Materials And Methods
Cell culture
Fluorescence in situ hybridization
Probes
Statistical analysis
Acknowledgements
References
REFERENCES
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