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Common fragile sites and genomic instability
Yuri Pekarsky, Alessandra Drusco, Eugenio Gaudio, Carlo M Croce, Nicola Zanesi
Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University,
Columbus, OH, USA (YP, AD, EG, CMC, NZ)
Published in Atlas Database: June 2013
Online updated version : http://AtlasGeneticsOncology.org/Deep/CommFragSitesID20122.html
DOI: 10.4267/2042/51877
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.
© 2013 Atlas of Genetics and Cytogenetics in Oncology and Haematology
induced by aphidicolin, an inhibitor of DNA synthesis
that, by affecting DNA polymerases alpha, delta and
epsilon, has been shown to activate most fragile sites
(Mrasek et al., 2010), inducing gaps that are
microscopically visible in metaphase chromosomes.
At the molecular level, the phenomenon of common
fragility on chromosomes is still not completely
understood (Brueckner et al., 2012). The ATR DNA
damage checkpoint pathway has been suggested to
have an important role in maintaining the stability of
CFSs since a deficiency of proteins associated with this
pathway, like ATR, BRCA1, and CHK1, results in
increased breakages of CFSs (Casper et al., 2002;
Durkin et al., 2008). Moreover, CFSs fragility has been
associated with late DNA replication (Debatisse et al.,
2006) and histone hypoacetylation (Jiang et al., 2009).
It has also been hypothesized that, following DNA
replication stress CFSs instability derives from
prolonged single-stranded regions of unreplicated DNA
accumulating at stalled replication forks that escaped
the ATR replication checkpoint (Brueckner et al.,
2012). In fact, some aCFSs with delayed late
replication due to aphidicolin treatment can enter G2
with only 50% of some aCFSs regions completely
replicated (Pelliccia et al., 2008). DNA breakage within
aCFSs is thought to derive from failing to complete
replication prior to the end of telophase and
chromosome segregation (Chan et al., 2009). It has
been recently shown that the activity of topoisomerase I
is necessary for CFSs fragility due to the requirement
for polymerase - helicase uncoupling (Arlt and Glover,
2010). It has been suggested that impaired replication
of such regions may be due to the formation of stable
secondary structures in their DNA sequences (Burrow
et al., 2010; Zlotorynski et al., 2003).
Keywords
CFSs, common fragile sites, aphidicolin, genomic
instability, FRA3B, FRA16D, CFS tumor suppressor
genes
General features
Specific alterations in the genome that modify the
expression of genetic elements involved in the
regulation of cell growth and maintenance of genomic
integrity are responsible for driving tumorigenesis.
These changes are not random, even though each tumor
has a particular set of genome alterations. Typically,
overexpression of oncogenes and inactivation of tumor
suppressor genes occur often and are being extensively
studied. Moreover, in malignant cells there is a group
of genomic loci that is frequently unstable and
contributes actively to tumorigenesis, the common
fragile sites (CFSs) (Casper et al., 2012). These regions
are non-random sites on chromosomes that under
conditions of DNA replication stress, such as mild
inhibition of DNA polymerase activity, form gaps and
breaks (Glover et al., 1984). As signified by the
"common" in their name, CFSs occur at specific
chromosome bands of all humans and are a normal
component of the chromosomal structure (Durkin et al.,
2008). These loci are conserved in other mammals,
including, but not limited to, primates and rodents
(Fungtammasan et al., 2012). Endogenous and
exogenous factors, such as hypoxia, chemotherapeutics
and other pharmaceuticals, exposure to radiations,
pesticides, cigarette smoke, caffeine and alcohol, may
trigger activation of replication fork stress and DNA
breaks at CFSs in vivo (Dillon et al., 2010). On the
other hand, in vitro, a subset of CFSs (aCFSs) may be
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Pekarsky Y, et al.
Table 1. Association of the best characterized common fragile sites with their chromosome regions and genes affected by their
activity. Modified from Saxena, 2012.
mechanism that determines the placement of replication
origins. This suggests that all the hypothesizing about
the effect that specific sequences have in fragile regions
may become questionable (Debatisse et al., 2012;
Huebner, 2011; Letessier et al., 2011).
While rare fragile sites are generally associated with a
single DNA element, several sequence motifs spread
along an aCFS locus may determine its fragility
(Durkin et al., 2008; Ragland et al., 2008) thus making
the characterization of aCFSs a computational
challenge (Fungtammasan et al., 2012). However,
previous analyses of single aCFSs showed that these
loci are enriched in Alu repeats (Tsantoulis et al.,
2008), gene-coding regions (Helmrich et al., 2006),
histone hypoacetylation (Jiang et al., 2009), high DNA
flexibility sequences, and highly AT-rich sequences
(Mishmar et al., 1998). Nevertheless, these sequence
characteristics seemed not to be associated with the
propensity for DNA gaps, breaks, deletions and other
genomic rearrangements at CFSs; for example, LINE1
elements are common in the fragile site FRA3B but
quite rare in FRA16D, while Alu repeats are dominant
in the latter (Ried et al., 2000).
The organization of human chromosomes was
traditionally investigated by a variety of banding
methods (Comings, 1978). Yunis and Soreng observed
that several types of fragile sites are more frequent in R
bands that have a relatively high gene and CpG island
density and correspond to early replicating genomic
regions (Yunis and Soreng, 1984).
Among different CFSs the level of fragility is variable
and the most fragile and bestcharacterized CFS in the
entire human genome is FRA3B at chromosome band
3p14.2 (Mrasek et al., 2010). The second and third
most active CFSs are FRA16D and FRAXB respectively
at 16q23.2 and Xp22.3. Generally, in somatic cells
CFSs are stable but in many cancers they display
frequent chromosomal aberrations. Lung, kidney,
breast, and digestive tract malignancies are mainly
where heterozygous and homozygous deletions are
Many of the CFS genomic loci have not yet been
molecularly defined. Thus far, the relatively well
characterized CFSs are the following: FRA1E, FRA2C,
FRA2G, FRA3B, FRA7G, FRA9G, FRA13A, FRA16D,
and FRAXB (Brueckner et al., 2012), summarized in
Table 1, that are all AT-dinucleotide-rich sites spanning
between 300 kb and 1 Mb (Schwartz et al., 2006).
Unlike rare fragile sites, in which fragility is
attributable to either CGG repeat expansions or ATrich minisatellites (Sutherland, 2003), in CFSs no such
long repeat motifs have been found. However, the nine
CFSs defined at molecular level seem to be
characterized by segments of discontinuous AT-rich
sequences potentially forming secondary structures
able to affect replication fork progression and thus
leading to chromosomal breakage (Dillon et al., 2010;
Zlotorynski et al., 2003). Accordingly, it has been
reported that specific DNA sequences, such as [A/T]n
and [AT/TA]n repeats, and/or the formation of non-B
DNA secondary structures within aCFSs can inhibit
replicative DNA polymerases (Shah et al., 2010) and
the progression of replication forks (Zhang and
Freudenreich, 2007). Recently, scarcity of replication
origins, inefficient origin initiation, and failure to
activate latent origins have all been proposed to play a
role in delayed replication at specific aCFSs (Letessier
et al., 2011; Ozeri-Galai et al., 2011).
The Debatisse Laboratory reported the most important
new findings in the recent years, showing that CFSs
differ in different tissue types and are caused by the
paucity of replication origins within the regions - i.e.
both FRA3B and FRA16D have replication origins
flanking the fragile locus and must replicate the DNA
from flanking sites to meet in the middle late S or in
G2, in lymphocytes, but the placement of replication
origins is different in fibroblasts and these loci are
much less fragile in fibroblasts, while different loci are
more fragile in fibroblasts. Obviously, this may apply
to other tissue types too and shows that the position of
fragile regions in specific tissues is due to an epigenetic
Atlas Genet Cytogenet Oncol Haematol. 2013; 17(12)
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Pekarsky Y, et al.
identified
as
the
most
common
genomic
rearrangements in CFSs (Arlt et al., 2006). All CFSs
investigated at molecular level up to now contain
protein-coding genes, most of which extend over
hundreds of kilobases of DNA (Smith et al., 2007). The
FHIT and WWOX genes encompassing FRA3B and
FRA16D, respectively, are both > 1 Mb in length and
have been shown to exhibit tumor suppressor activity in
vivo and in vitro (Drusco et al., 2011; Lewandowska et
al., 2009; Saldivar et al., 2010). There are many reports
of deletions within CFSs harboring these genes
(McAvoy et al., 2007). Actually, the fact that very large
genes present in mammalian genomes are preferentially
affected by deletions in tumor cells suggests that these
genes are all CFSs in the cell type in which they are
expressed (Debatisse et al., 2012). Mitotic sister
chromatid exchanges are often described at CFSs
(Durkin et al., 2008), which suggests that CFS breaks
may possibly drive loss of heterozygosity (LOH) in
cancer cells when the repair occurs by homologous
recombination.
During neoplastic progression, damage at CFS regions
seems to be among the earliest occurrences, mainly due
to DNA replication stress (Halazonetis et al., 2008) as
suggested by the presence of these genomic alterations
in pre-neoplastic lesions (Lai et al., 2010). Oncogene
amplification and preferred integration sites for some
oncogenic viruses are also triggered by CFS activity
(Brueckner et al., 2012).
Germline genomic alterations in CFSs seem also to
lead to other human illnesses of nonmalignant origin.
In support of this possibility is the recent sequencing of
breakpoint junctions in the CFS genes PARK2 at
FRA6E and DMD at FRAXC in many patients affected,
respectively, by juvenile Parkinsonism and muscular
dystrophies (Mitsui et al., 2010). Somatic breakpoints
in cancer cell lines and germline breakpoints within
PARK2 and DMD shared some features that suggested
involvement of common mechanisms in the generation
of CFS rearrangements.
that take place within domains spatially and temporally
separated (Wei et al., 1998). Usually transcription
occurs in G1 phase and sometimes in S phase.
When this happens, transcription is thought to be
spatially separated from replication sites (Vieira et al.,
2004). Gene expression induction in mammalian cells
caused
recombination
processes
within
the
transcription unit, thus suggesting that collisions
between replication and transcription complexes
provoke instability at the genomic level (Gottipati et
al., 2008). Recently, Helmrich et al. demonstrated that
the time required to transcribe human genes larger than
800 kb spans more than one complete cell cycle, while
their transcription speed is equivalent to that of smaller
genes. CFS instability depends on the expression of the
underlying long genes and may be suppressed by
RNase H1 enzyme when intervenes on R-loops, which
are RNA:DNA hybrids between nascent transcripts and
the DNA template strand, while the nontemplate strand
remains as single-stranded DNA (Helmrich et al.,
2011).
The wealth of genome-wide profiling studies now
available offers unique opportunities to study causes of
genome instability in depth. Current evidence suggests
that aCFSs are caused by a series of genomic factors
(Dillon et al., 2010). Consequently, building a
statistical model that takes into consideration multiple
factors simultaneously is thought to be more
biologically reliable on the contribution to fragility by
the diverse genomic features. Moreover, studies usually
do not incorporate in their models the different
breakage frequencies of aCFSs.
To better understand the relationship between aCFSs
and their genomic contexts, Fungtammasan et al. built
statistical models to explain the fragility of wellcharacterized aCFSs by considering their genomic
neighborhoods and comparing them with non-fragile
regions (NFRs) (Fungtammasan et al., 2012).
The authors focused on aphidicolin-induced CFSs
because they are well-characterized genomewide
(Mrasek et al., 2010), are the most numerous CFSs, and
fragile sites induced by other agents might have
different breakage mechanisms and characteristics.
Multiple logistic regression was used to predict the
probability of a given region to be either an aCFS or an
NFR and multiple linear regression for the prediction of
expected breakage frequency. Eventually these models
were
validated
using
mouse
fragile
sites
(Fungtammasan et al., 2012). Results showed that local
genomic features are effective predictors both of
regions harboring aCFSs, explaining circa 77% of the
deviance in logistic regression models, and of aCFS
breakage frequencies, explaining approximately 45% of
the variance in standard regression models.
In models with the highest explanatory power, aCFSs
are mainly located in G-negative chromosomal bands
and far from centromeres, are enriched in Alu repeats,
and have high DNA flexibility. In addition, aCFSs have
Recent developments
DNA replication and gene transcription are basic
biological processes essential for cell division and
growth. Large protein complexes moving at high speed
along the chromosomes, and for long distances, make
such processes possible. The RNA polymerase II (Pol
II) enzyme, in mammalian cells, transcribes 18-72
nucleotides of DNA per second into RNA (Darzacq et
al., 2007). One of the longest human loci, the 2.2 Mb
dystrophin gene, is transcribed over a period of 16
hours (Tennyson et al., 1995) and similar figures are
reported for other long genes. As for typical fastcycling mammalian cells the cell cycle time is
approximately 10 hours, it is expected that these longterm transcription cycles interfere with replication in
cell cycle S phase. Unlike in bacteria, transcription and
replication in higher eukaryotes are coordinated events
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Common fragile sites and genomic instability
Pekarsky Y, et al.
Fhit+/+ cells (Turner et al., 2002), and the frequency
of mutations following replicative and oxidative stress
in Fhit-deficient cells was 2 to 5-fold greater than in
Fhit-expressing cells (Ishii et al., 2008; Ottey et al.,
2004). Despite these findings and strong evidence that
Fhit acts as a tumor suppressor (Joannes et al., 2010;
Pekarsky et al., 1998; Siprashvili et al., 1997) it has
been proposed that deletions within the FHIT locus are
secondary alterations rather than cancer-driving
mutations (Bignell et al., 2010). In a new study, Kay
Huebner and colleagues (Saldivar et al., 2012)
examined further the role of Fhit loss in DNA damage
process. Specifically, it has been shown that Fhit loss
causes replication stress-induced DNA double-strand
breaks in normal, transformed, and cancer-derived cell
lines. In Fhit-deficient cells, a defect was observed in
replication fork progression that stemmed mainly from
fork stalling and collapse. The possible mechanism for
the role of Fhit in replication fork progression is by
regulation of thymidine kinase 1 expression and
thymidine triphosphate pool levels. Interestingly,
restoration of nucleotide balance rescued DNA
replication defects and suppressed DNA breakage in
Fhit-deficient cells. Loss of Fhit did not activate the
DNA damage response nor cause cell cycle arrest,
allowing continued cell proliferation and ongoing
chromosome instability. Such a result was consistent
with in vivo studies, where Fhit knockout mouse tissues
showed no evidence of cell cycle arrest or senescence
yet exhibited numerous somatic DNA copy number
aberrations at replication-sensitive loci. Moreover, cells
established from Fhit KO tissues showed rapid
immortalization together with DNA deletions and
amplifications. Of note, the murine gene Mdm2, an
oncogene involved in cell transformation, was also
amplified with 4-fold increase in Mdm2 mRNA
expression, suggesting that genome instability induced
by FHIT depletion facilitates the transformation
process. In conclusion, this study proposes that Fhit
depletion in precancerous lesions is the first step in the
initiation of genomic instability and links alterations at
CFSs to the very origin of this important phenomenon
(Saldivar et al., 2012).
To conclude this short panoramic on CFSs and
genomic instability, we would like to draw the reader's
attention to the most recent findings about Polζ
polymerase. Polζ, which consists of the catalytic
subunit Rev3 and the accessory subunit Rev7, is a
trans-lesion DNA synthesis (TLS) polymerase capable
of bypassing certain DNA adducts efficiently (Gibbs et
al., 1998). Besides its role in TLS, Rev3 is also
essential for mouse embryonic development (Bemark et
al., 2000), whereas no other TLS polymerases studied
to date are required for this fundamental function. Rev3
has been also implicated in homologous recombination
repair (Sharma et al., 2012). Because of its extremely
large size (>350 kDa), little progress has been made in
understanding the essential function of Rev3. Bhat et
high fragility when co-located with evolutionarily
conserved chromosomal breakpoints (Fungtammasan et
al., 2012).
In order to investigate the mechanisms of CFS-induced
breaks, Casper et al. asked whether the flexibility peaks
that have been identified within human CFS FRA3B
are hotspots of instability (Casper et al., 2012). These
authors, to analyze the consequences of CFS breaks,
also investigated whether repair of fragile site breaks
drives LOH events due to mitotic homologous
recombination. To gather detailed data on exact break
locations within CFSs, a yeast artificial chromosome
(YAC) containing the human locus FRA3B was used.
Data suggested that break sites are not randomly
distributed, but rather clustered at the centromere-distal
end of the FRA3B sequence insert. They also took
advantage of a naturally occurring yeast fragile site
known as FS2 (fragile site 2) to study mitotic
homologous recombination. Similar to human CFSs,
recurrent breaks at FS2 occur where replication is
impaired because of stressful conditions (Lemoine et
al., 2005). Results demonstrated that LOH is, in fact, a
consequence of mitotic recombination between
homologous chromatids with reciprocal crossovers at
FS2 induced by inhibition of yeast DNA polymerase
(Casper et al., 2012). Since not many CFSs have been
molecularly characterized, despite the growing interest
in understanding the precise nature of CFS instability,
Brueckner et al. took into consideration the FRA2H
CFS and after having fine-mapped the location with
six-color
fluorescence
in
situ
hybridization,
demonstrated that it is one of the most active CFSs in
the human genome (Brueckner et al., 2012). FRA2H
encompasses approximately 530 kb of a gene-poor
region containing a novel large inter-genic non coding
RNA gene (AC097500.2). Using custom-designed
array comparative genomic hybridization, gross and
submicroscopic chromosomal rearrangements were
detected, involving FRA2H in a panel of 54
neuroblastoma, colon, and breast cancer cell lines.
Genomic alterations often affected different classes of
long terminal repeats (LTRs) and long interspersed
nuclear elements (LINEs). Sequence analysis of
breakpoint junctions revealed that DNA damage repair
at FRA2H mostly appeared to occur via nonhomologous end-joining events mediated by short
micro-homologies (Brueckner et al., 2012).
Deletions at FRA3B CFS occur in pre-neoplasias and
may be the most frequent and earliest alterations.
FRA3B overlaps the FHIT gene, and its fragility
frequently results in deletions of FHIT exons and loss
of FHIT expression in precancerous and cancer cells
(Sozzi et al., 1998). Examination of cells that have lost
FHIT revealed that the protein has some functional
roles in response to DNA damage (Saldivar et al.,
2010). In particular, kidney epithelial cells established
from Fhit-/- mice exhibited >2-fold increased
chromosome breaks at fragile sites vs. corresponding
Atlas Genet Cytogenet Oncol Haematol. 2013; 17(12)
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Common fragile sites and genomic instability
Pekarsky Y, et al.
CM, Pilotti S. Loss of FHIT function in lung cancer and
preinvasive bronchial lesions. Cancer Res. 1998 Nov
15;58(22):5032-7
al. found that the cellular level of Rev3 is elevated in
mitotic cells, and the protein is associated with
chromatin. Experimental depletion of Rev3 results in
elevated CFS expression and chromosomal instability,
indicating that Rev3 is required for the late replication
of these sites. Rev3 activity is independent of Rev7, as
the depletion of cellular Rev7 does not cause CFS
expression. Moreover, constitutive depletion of Rev3 in
cultured human cells resulted in accumulated genomic
instability and eventual arrest of cell division,
suggesting that Rev3 is required not only for embryonic
development but also for cell viability (Bhat et al.,
2013). Interestingly, comparison of yeast and
mammalian Rev3 proteins reveals a large exon that is
unique to the mammalian gene that will surely be
subjected to future investigations for its role in the
maintenance of mitotic genomic stability.
Wei X, Samarabandu J, Devdhar RS, Siegel AJ, Acharya R,
Berezney R. Segregation of transcription and replication sites
into
higher
order
domains.
Science.
1998
Sep
4;281(5382):1502-6
Bemark M, Khamlichi AA, Davies SL, Neuberger MS.
Disruption of mouse polymerase zeta (Rev3) leads to
embryonic lethality and impairs blastocyst development in vitro.
Curr Biol. 2000 Oct 5;10(19):1213-6
Ried K, Finnis M, Hobson L, Mangelsdorf M, Dayan S,
Nancarrow JK, Woollatt E, Kremmidiotis G, Gardner A, Venter
D, Baker E, Richards RI. Common chromosomal fragile site
FRA16D sequence: identification of the FOR gene spanning
FRA16D and homozygous deletions and translocation
breakpoints in cancer cells. Hum Mol Genet. 2000 Jul
1;9(11):1651-63
Casper AM, Nghiem P, Arlt MF, Glover TW. ATR regulates
fragile site stability. Cell. 2002 Dec 13;111(6):779-89
Acknowledgements
Turner BC, Ottey M, Zimonjic DB, Potoczek M, Hauck WW,
Pequignot E, Keck-Waggoner CL, Sevignani C, Aldaz CM,
McCue PA, Palazzo J, Huebner K, Popescu NC. The fragile
histidine triad/common chromosome fragile site 3B locus and
repair-deficient cancers. Cancer Res. 2002 Jul 15;62(14):405460
We thank Dr. Kay Huebner for critical reading of the
manuscript and Prasanthi Kumchala for technical
assistance. This work was supported by NIH grant
U01CA152758 (to CMC).
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Pekarsky Y, Drusco A, Gaudio E, Croce CM, Zanesi N.
Common fragile sites and genomic instability. Atlas Genet
Cytogenet Oncol Haematol. 2013; 17(12):849-855.
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