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Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis. This mitotic DNA synthesis, termed MiDAS, requires the MUS81-EME1 endonuclease and a non-catalytic subunit of the Pol-delta complex, POLD3. Here, we examine the contribution of HR factors in promoting MiDAS in human cells. We report that RAD51 and BRCA2 are dispensable for MiDAS but are required to counteract replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective inhibition of MiDAS may comprise a potential therapeutic strategy to sensitize cancer cells undergoing replicative stress.

Common fragile sites (CFSs) correspond to chromosomal regions susceptible to present breaks, discontinuities or constrictions in metaphase chromosomes from cells subjected to replication stress. They are considered as genomic regions intrinsically difficult to replicate and they are evolutionary conserved at least in mammals. However, the recent discovery that CFSs are cell-type specific indicates that DNA sequence by itself cannot account for CFS instability. Nevertheless, the large gene FHIT that includes FRA3B, the most highly expressed CFS in human lymphocytes, is commonly deleted in a variety of tumors suggesting a tumor suppressor role for its product. Here, we report that the epicenter of fragility of Fra14A2/Fhit, the mouse ortholog of human FRA3B/FHIT that like its human counterpart is the most highly expressed CFS in mouse lymphocytes, is largely attached to the nuclear matrix compartment in naive B lymphocytes but not in primary hepatocytes or cortical neurons that do not express such a CFS. Our results suggest a structural explanation for the difficult-to-replicate nature of such a region and so for its common fragility in lymphocytes, that is independent of the possible tumor suppressor role of the gene harboring such CFS.

A conspicuous cell-shape phenotype known as &ldquo, screwy&rdquo, was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5&ndash, 6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32’s role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism., Author summary Cancer etiological studies suggest that the majority of pathological mutations occurred under near normal DNA replication conditions, emphasizing the importance of understanding replication regulation under non-lethal conditions. To gain such a better understanding, we investigated the function of Pol32, a conserved ancillary subunit of the essential DNA polymerase Delta complex, through the development of the fruit fly Drosophila. We uncovered a previously unappreciated function of Pol32 in regulating the nuclear import of the polymerase complex, and this function is developmentally regulated. By utilizing mutations in pol32 and other replication factors, we have started to define basic features of Chromosome Fragile Sites (CFS) in Drosophila somatic cells. CFS is a major source of genome instability associated with replication stresses, and has been an important topic of cancer biology. We discovered that CFS formation does not favor genomic regions with repetitive sequences except the highly transcribed locus encoding ribosomal RNA. Our work lays the groundwork for future studies using Drosophila as an alternative system to uncover the most fundamental features of CFS.

Some regions of the genome, notably common fragile sites (CFSs), are hypersensitive to replication stress and often involved in the generation of gross chromosome rearrangements in cancer cells. CFSs nest within very large genes and display cell-type-dependent instability. Fragile or not, large genes tend to replicate late in S-phase. A number of data now show that transcription perturbs replication completion across the body of large genes, particularly upon replication stress. However, the molecular mechanisms by which transcription elicits such under-replication and subsequent instability remain unclear. We present here our view of the mechanisms responsible for CFS under-replication and those allowing the cells to cope with this problem in G2 and mitosis. We notably focus on the major role played by the FANC proteins in the protection of CFSs from S phase up to late mitosis. We finally discuss a possible rationale for the conservation of large genes across vertebrate evolution.

Amongst 15 bird species, representative of 7 orders, recurrent breakages evocating the presence of fragile sites were detected in the chromosomes of the 5 species belonging to Passeriformes. These breaks appeared when 5-bromodeoxyuridine (BrdU) was added to the cell culture medium at a dose inefficient for inducing chromosome structure alterations in other birds and mammals. They involved, similarly in male and female, 3 loci on the Z chromosome of 3 Turdus species (Turdidae). Labeling by BrdU antibody confirmed the correlation between BrdU incorporation into DNA and breakage, especially around and in the sites of breakage. Thus, 3 BrdU-sensitive fragile sites were present in the Z chromosomes of these birds. Three fragile sites were also detected at different locations in the Z chromosomes of the European robin (Erithacus rubecula, Muscicapidae), suggesting that a structural rearrangement occurred during the evolution of Turdidae and Muscicapidae. Chromosome banding confirmed this interpretation. Finally, in the more distantly related species Parus major (Paridae), the almost acrocentric Z chromosome displayed a single BrdU-sensitive fragile site in its short arm, and the W appeared to be pulverized by BrdU incorporation. Although it cannot be excluded that the BrdU-sensitive fragile sites may be involved in rearrangements, their conservation in many species, and possibly all Passeriformes, provides evidence that they do not constitute a pejorative character during evolution.

Evolution generates a remarkable breadth of living forms, but many traits evolve repeatedly, by still poorly understood mechanisms. A classic example of repeated evolution is the loss of pelvic hindfins in stickleback fish (Gasterosteus aculeatus). Repeated pelvic loss maps to recurrent deletions of a pelvic enhancer of the Pitx1 gene. Here, we identify molecular features contributing to these recurrent deletions. Pitx1 enhancer sequences form alternative DNA structures in vitro, and increase double-strand breaks and deletions in vivo. Enhancer mutability depends on DNA replication direction and is caused by (TG)-dinucleotide repeats. Modeling shows that elevated mutation rates can influence evolution under demographic conditions relevant for sticklebacks and humans. DNA fragility may thus help explain why the same loci are often used repeatedly during parallel adaptive evolution.

Summary Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication. Fragility at Flex1 is initiated by structure-specific endonuclease Mus81-Mms4 acting together with the Slx1-4/Rad1-10 complex, whereas Yen1 protects Flex1 against breakage. Sae2 is required for healing of Flex1 after breakage. Our study shows that breakage within a CFS can be initiated by nuclease cleavage at forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also are impaired in their ability to heal, and this deleterious combination accounts for their fragility.

Collision between the molecular machineries responsible for transcription and replication is an important source of genome instability. Certain transcribed regions known as chromosomal fragile sites are particularly prone to recombine and mutate in a manner that correlates with specific transcription and replication patterns. At the same time, these chromosomal fragile sites engage in aberrant DNA structures in mitosis. Here, we discuss the mechanistic details of transcription-replication conflicts including putative scenarios for R-loop-induced replication inhibition to understand how transcription-replication conflicts transition from S phase into various aberrant DNA structures in mitosis.

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5'-part from the 3'-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms.

Common fragile sites (CFSs) are chromosomal regions characterized as hotspots for breakage and chromosomal rearrangements following DNA replication stress. They are preferentially unstable in pre-cancerous lesions and during cancer development. Recently CFSs were found to be tissue- and even oncogene-induced specific, thus indicating an unforeseen complexity. Here we review recent developments in CFS research that shed new light on the molecular basis of their instability and their importance in cancer development.

Sites of 45S rDNA of Lolium are regions denominated fragile sites (FSs), constituting regions slightly stained with DAPI due to increased DNA unpacking in metaphasic chromosomes. Considered to be fragile regions in the genome, the FSs might be more responsive to induced breaks and result in chromosomal fragments and rearrangements, unless repairing mechanisms such as recombination or de novo telomere formation play a role at the break site of the DNA. Thus, this study aimed at investigating if SFs from Lolium are hotspots for the occurrence of breakages induced by X-ray and if they are regions favorable to synthesize new telomeres, using Hordeum vulgare as a comparative model. Lolium multiflorum and H. vulgare seedlings were irradiated with 20 and 50 Gy X-ray and evaluated one day following the irradiation and at 7-days intervals for a 28-days period, using FISH technique with 45S rDNA and Arabidopsis-type telomere probes in order to investigate the presence of chromosomal breakages and new telomere formation. H. vulgare did not survive after a few days of irradiation due to the increased rate of abnormalities. L. multiflorum also exhibited chromosomal abnormalities following the exposure, yet over the 28-days trial it had a decrease in the chromosomal damage rate and formation of de novo telomere has not been detected along this time. Despite being considered to be fragile regions in the genome, the 45S rDNA sites of Lolium are not hotspots to chromosomal breakages after the induction of breakages.

The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATMflox) was crossed to hypomorphic NBS1 mutants (Nbs1ΔB/ΔB mice). Nbs1ΔB/ΔB Atm−/− hematopoietic cells derived by crossing to vavcre were viable in vivo. Nbs1ΔB/ΔB Atm−/− VAV mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. Implications: A novel mouse model was developed, by combining a vavcre-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis. Mol Cancer Res; 14(2); 185–95. ©2015 AACR.

FHIT, located at FRA3B, is one of the most commonly deleted genes in human cancers, and loss of FHIT protein is one of the earliest events in cancer initiation. However, location of FHIT at a chromosomal fragile site, a locus prone to breakage and gap formation under even mild replication stress, has encouraged claims that FHIT loss is a passenger event in cancers. We summarize accumulated evidence that FHIT protein functions as a genome “caretaker” required to protect the stability of genomes of normal cells of most tissues from agents causing intrinsic and extrinsic DNA damage. FHIT loss leads to intracellular replication stress and subsequent genome instability, which provides an opportunistic mutational landscape in preneoplasias for selection of a variety of other cancer-driving mutations. We also review evidence showing that FHIT loss leads to enhanced activation of other common fragile sites, including the FRA16D/WWOX locus, and creates optimal single-stranded DNA substrates for the hypermutator enzyme, APOBEC3B.

Data published in the scientific literature suggests a possible link between chromosomal rearrangements involving autosomes 1 and 3 and the presence of red morphs in the peach-potato aphid Myzus persicae (Sulzer). In order to begin a study of this relationship, we analysed the genomic and chromosomal location of genes involved in carotenoid biosynthesis in M. persicae and the pea aphid, Acyrthosiphon pisum (Harris), since carotenoids are the basis of the colour in many aphid species. Genomic analysis identified a DNA sequence containing carotenoid genes in synteny between the 2 species. According to the results obtained using in situ PCR, carotenoid genes were located in a subterminal portion of autosome 1 in both species. The same localization has also been observed in the onion aphid Neotoxoptera formosana Takahashi that, as M. persicae and A. pisum, belongs to the tribe Macrosiphini, thereby suggesting a synteny of this chromosomal region in aphids. In situ PCR experiments performed on 2 M. persicae asexual lineages bearing heterozygous translocations involving autosomes 1 and 3 revealed that carotenoid genes were located within chromosomal portions involved in recurrent rearrangements. We also verified by bioinformatics analyses the presence of fragile sites that could explain these recurrent rearrangements in M. persicae.

The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi,P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution. Fil: Suarez, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Ferro, Juan Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Nagamachi, Cleusa Y.. Universidade Federal do Pará; Brasil Fil: Cardozo, Dario Elbio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Blasco zuñiga, Ailin. Pontificia Universidad Católica del Ecuador; Ecuador Fil: Silva, Jéssica B.. Universidade Federal do Pará; Brasil Fil: Marciano Jr, Euvaldo. Universidade Estadual de Santa Cruz; Brasil Fil: Costa, Marco A.. Universidade Estadual de Santa Cruz; Brasil Fil: Orrico, Victor G.D.. Universidade Estadual de Santa Cruz; Brasil Fil: Solé, Mirco. Universidade Estadual de Santa Cruz; Brasil Fil: Roberto, Igor J.. Universidad Federal del Amazonas.; Brasil Fil: Rivera, Miryan. Pontificia Universidad Católica del Ecuador; Ecuador Fil: Wiley, John E.. University School of Medicine; Estados Unidos Fil: Faivovich, Julián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina Fil: Baldo, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Pieczarka, Julio. Universidade Federal do Pará; Brasil

DNA Helicase B (HELB) is a conserved helicase in higher eukaryotes with roles in the initiation of DNA replication and in the DNA damage and replication stress responses. HELB is a predominately nuclear protein in G1 phase where it is involved in initiation of DNA replication through interactions with DNA topoisomerase 2-binding protein 1 (TOPBP1), cell division control protein 45 (CDC45), and DNA polymerase α-primase. HELB also inhibits homologous recombination by reducing long-range end resection. After phosphorylation by cyclin-dependent kinase 2 (CDK2) at the G1 to S transition, HELB is predominately localized to the cytosol. However, this cytosolic localization in S phase is not exclusive. HELB has been reported to localize to chromatin in response to replication stress and to localize to the common fragile sites 16D (FRA16D) and 3B (FRA3B) and the rare fragile site XA (FRAXA) in S phase. In addition, HELB is phosphorylated in response to ionizing radiation and has been shown to localize to chromatin in response to various types of DNA damage, suggesting it has a role in the DNA damage response.

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity., Author summary Increasing evidence suggest that instabilities at common fragile sites (CFSs), breakage-prone genomic loci, may be source of genomic aberration seen in cancer cells. Among the proposed mechanisms that can cause CFSs instabilities is the conflict between transcription and replication, and the mechanisms or factors that resolve the possible conflicts are only beginning to be understood. Here we found that deficiency in the Polycomb group proteins BMI1 or RNF2 leads to the CFS instability, and is associated with transcription-associated replication fork stresses. We further found that in the absence of RNF2, cells depend on the Fanconi Anemia fork-protective proteins for genome maintenance and survival. These results underscore that the Polycomb proteins are important for genome maintenance.

Alternative non-B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single-stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT-rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance.

Mechanisms and events related to common fragile site (CFS) instability are well known in cancer cells. Here, we argue that normal cells remain an important experimental model to address questions related to CFS instability in the absence of alterations in cell cycle and DNA damage repair pathways, which are common features acquired in cancer. Furthermore, a major gap of knowledge concerns the stability of CFSs during gametogenesis. CFS instability in meiotic or postmeiotic stages of the germ cell line could generate chromosome deletions or large rearrangements. This in turn can lead to the functional loss of the several CFS-associated genes with tumor suppressor function. Our hypothesis is that such mutations can potentially result in genetic predisposition to develop cancer. Indirect evidence for CFS instability in human germ cells has been provided by genomic investigations in family pedigrees associated with genetic disease. The issue of CFS instability in the germ cell line should represent one of the future efforts, and may take advantage of the existence of sequence and functional conservation of CFSs between rodents and humans.

Common fragile sites (CFSs) are specific genomic regions in normal chromosomes that exhibit genomic instability under DNA replication stress. As replication stress is an early feature of cancer development, CFSs are involved in the signature of genomic instability found in malignant tumors. The landscape of CFSs is tissue-specific and differs under different replication stress inducers. Nevertheless, the features underlying CFS sensitivity to replication stress are shared. Here, we review the events generating replication stress and discuss the unique characteristics of CFS regions and the cellular responses aimed to stabilizing these regions.

Common fragile sites (cFSs) represent parts of the normal chromosome structure susceptible to breakage under replication stress. Although only a small number of cFSs have been molecularly characterized, genomic damage of cFS genes appears to be critical for the development of various human diseases. In this study, we fine mapped the location of FRA14B and showed that the fragile region spans 765 kb at 14q23.3, containing the large gephyrin (GPHN) gene. The FRA14B sequence is enriched in perfect A/T>24 stretches and R-loop forming sequences (RLFS), and harbors a large palindromic motif in the core region. FRA14B instability is not only limited to lymphocytes, but also occurs in neuroblastoma and breast epithelial cells. Using array comparative genomic hybridization (CGH), we examined copy number alteration patterns within FRA14B in a panel of 180 cancer cell lines and primary tumors. Our CGH data and a survey of 1046 Cancer Cell Line Encyclopedia profiles demonstrate that focal deletions cluster within FRA14B and disrupt the genomic integrity of GPHN in approximately 5% of cancer cells. Moreover, germline CNVs (copy number variants) profiles provided by the Database of Genomic Variants and available literature suggest that germline CNVs and rare pathogenic deletions associated with neurodevelopmental disorders cluster within the core fragile region of GPHN. Overall, our data provide insight into the molecular structure of FRA14B, and identify GPHN, as a large cFS gene in the human genome, whose disruption appears to trigger various neurodevelopmental diseases.

The selective pressures that shape clonal evolution in healthy individuals are largely unknown. Here we investigate 8,342 mosaic chromosomal alterations, from 50 kb to 249 Mb long, that we uncovered in blood-derived DNA from 151,202 UK Biobank participants using phase-based computational techniques (estimated false discovery rate, 6–9%). We found six loci at which inherited variants associated strongly with the acquisition of deletions or loss of heterozygosity in cis. At three such loci (MPL, TM2D3–TARSL2, and FRA10B), we identified a likely causal variant that acted with high penetrance (5–50%). Inherited alleles at one locus appeared to affect the probability of somatic mutation, and at three other loci to be objects of positive or negative clonal selection. Several specific mosaic chromosomal alterations were strongly associated with future haematological malignancies. Our results reveal a multitude of paths towards clonal expansions with a wide range of effects on human health.

MicroRNAs (miRNAs) are a class of short non-coding RNAs (ncRNAs) with typical sequence lengths of 19-25 nucleotides and extraordinary abilities to regulate gene expression. Because miRNAs regulate multiple important biological functions of the cell (proliferation, migration, invasion, apoptosis, differentiation, and drug resistance), their expression is highly controlled. Genetic and epigenetic alterations frequently found in cancer cells can cause aberrant expression of miRNAs and, consequently, of their target genes. The tumor microenvironment can also affect miRNA expression through soluble factors (e.g., cytokines and growth factors) secreted by either tumor cells or non-tumor cells (such as immune and stromal cells). Furthermore, like hormones, miRNAs can be secreted and regulate gene expression in recipient cells. Altered expression levels of miRNAs in cancer cells determine the acquisition of fundamental biological capabilities (hallmarks of cancer) responsible for the development and progression of the disease.

Purpose: Using genome-wide noninvasive prenatal screening (NIPS), we detected a 20-megabase specific deletion starting at 10q25 in eight pregnancies. The deletion could not be confirmed by invasive testing. Since all 10(q25→qter) deletions started close to the FRA10B fragile site in 10q25, we investigated whether the pregnant women were indeed carriers of FRA10B. Methods: We performed NIPS analysis for all autosomes using single-read sequencing. Analysis was done with the WISECONDOR algorithm. Culture of blood lymphocytes with bromodeoxyuridine was used to detect FRA10B expansions. Fluorescence in situ hybridization and array analysis were used to find maternal and/or fetal deletions. Results: We confirmed the presence of a FRA10B expansion in all four tested mothers. Fluorescence in situ hybridization and array analysis confirmed the presence of a maternal mosaic deletion of 10(q25→qter). Conclusion: The recurring 10(q25→qter) deletion detected with NIPS is a false-positive result caused by a maternal low-level mosaic deletion associated with FRA10B expansions. This has important consequences for clinical follow-up, as invasive procedures are unnecessary. Expanded maternal FRA10B repeats should be added to the growing group of variants in the maternal genome that may cause false-positive NIPS results.

Background Fragile sites are the chromosomal regions that are susceptible to breakage, and their frequency varies among the human population. Based on the frequency of fragile site induction, they are categorized as common and rare fragile sites. Common fragile sites are sensitive to replication stress and often rearranged in cancer. Rare fragile sites are the archetypal trinucleotide repeats. Fragile sites are known to be involved in chromosomal rearrangements in tumors. Human miRNA genes are also present at fragile sites. A better understanding of genes and miRNAs lying in the fragile site regions and their association with disease progression is required. Result HumCFS is a manually curated database of human chromosomal fragile sites. HumCFS provides useful information on fragile sites such as coordinates on the chromosome, cytoband, their chemical inducers and frequency of fragile site (rare or common), genes and miRNAs lying in fragile sites. Protein coding genes in the fragile sites were identified by mapping the coordinates of fragile sites with human genome Ensembl (GRCh38/hg38). Genes present in fragile sites were further mapped to DisGenNET database, to understand their possible link with human diseases. Human miRNAs from miRBase was also mapped on fragile site coordinates. In brief, HumCFS provides useful information about 125 human chromosomal fragile sites and their association with 4921 human protein-coding genes and 917 human miRNA’s. Conclusion User-friendly web-interface of HumCFS and hyper-linking with other resources will help researchers to search for genes, miRNAs efficiently and to intersect the relationship among them. For easy data retrieval and analysis, we have integrated standard web-based tools, such as JBrowse, BLAST etc. Also, the user can download the data in various file formats such as text files, gff3 files and Bed-format files which can be used on UCSC browser. Database URL: http://webs.iiitd.edu.in/raghava/humcfs/ Electronic supplementary material The online version of this article (10.1186/s12864-018-5330-5) contains supplementary material, which is available to authorized users.

Massive genomic rearrangements, a result of single catastrophic event termed chromothrispsis or chromosomal catastrophe, have been identified in a variety of human cancers. In a few cancer types, chromothripsis was found to be associated with poor prognosis. We performed mate-pair sequencing and analysis of structural rearrangements in 132 prostate cancer cases which included clinically insignificant Gleason score 6 tumors, clinically significant tumors of higher grade (7+) and high grade prostatic intraepithelial neoplasia. Chromothripsis was observed at least 30 per cent of the samples across different grades. Surprisingly, it was frequently observed in clinically insignificant Gleason score 6 tumors, indicating that chromothripsis does not define more aggressive phenotype. The degree of chromothripsis did not increase significantly in tumors of advanced grades and did not appear to contribute to tumor progression. Our data showed that distribution of chromothriptic rearrangements differed from that of fragile sites but correlated with the size of chromosomes. We also provided evidence that rearrangements resulting from chromothripsis were present in the cells of neighboring Gleason patterns of the same tumor. Our data suggest that that chromothripsis plays role in prostate cancer initiation.

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS. © 2015 Wiley Periodicals, Inc.

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity. A.J.L.-C. and C.A. were funded a post-doctoral fellowship from the Spanish Association for Cancer Research (AECC). J.S. is a recipient of a predoctoral fellowship from the Spanish government (BES-2012-05 2030). S.V. and H.G. are funded by the Swedish Research Council and the Swedish Cancer Society. Work in O.F.-C.'s laboratory was supported by Fundacion Botin, Banco Santander through its Santander Universities Global Division, and grants from Ministerio de Economia y Competitividad (MINECO; SAF2011-23753), Worldwide Cancer Research (12-0229), Fundacio La Marato de TV3, Howard Hughes Medical Institute, and the European Research Council (ERC-617840). Work in A.J.L.-C.'s laboratory is funded by the Danish Council for Independent Research (DFF) and the Danish National Research Foundatio Sí

Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.

The WWOX gene spans the common chromosomal fragile site FRA16D that is located within a massive (780 kb) intron. The WWOX gene is very long, at 1.1 Mb, which may contribute to the very low abundance of the full-length 1.4 kb mRNA. Alternative splicing also accounts for a variety of aberrant transcripts, most of which are devoid of C-terminal sequences required for WWOX to act as an oxidoreductase. The mouse WWOX gene also spans a chromosomal fragile site implying some sort of functional relationship that confers a selective advantage. The encoded protein domains of WWOX are conserved through evolution (between humans and Drosophila melanogaster) and include WW domains, an NAD -binding site, short-chain dehydrogenase/reductase enzyme and nuclear compartmentalization signals. This homology has enabled functional analyses in D. melanogaster that demonstrate roles for WWOX in reactive oxygen species regulation and metabolism. Indeed the human WWOX gene is also responsive to altered metabolism. Cancer cells typically exhibit altered metabolism (Warburg effect). Many cancers exhibit FRA16D DNA instability that results in aberrant WWOX expression and is associated with poor prognosis for these cancers. It is therefore thought that aberrant WWOX expression contributes to the altered metabolism in cancer. In addition, others have found that a specific (low-expression) allele of WWOX genotype contributes to cancer predisposition.

SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational repair. We have also shown that deficiency of POLD3 leads to an increase in RPA-bound ssDNA when cells are under replication stress, suggesting that POLD3 plays a role in the cellular response to DNA replication stress. Considering that DNA replication stress is a source of genome instability, and that excessive replication stress is a hallmark of pre-neoplastic and tumor cells, our characterization of SUMO2 targets during a perturbed S-phase should provide a valuable resource for future functional studies in the fields of DNA metabolism and cancer biology.

Copy number variants (CNVs) of the Williams-Beuren syndrome (WBS) 7q11.23 region are responsible for neurodevelopmental disorders with multisystem involvement and variable expressivity. We found 2 patients with a deletion and 1 patient with a duplication in this region sharing a common breakpoint located between the LIMK1 and EIF4H(WBSCR1) genes. One patient had a WBS phenotype, although testing with a commercially available FISH assay was negative for the deletion. A further test using array CGH showed an atypical WBS region deletion. The second patient showed global developmental delay, speech delay and poor motor skills with a deletion outside the WBS region. The third patient had manifestations compatible with an autism spectrum disorder showing a duplication in the WBS region. Our findings point to the existence of a previously unrecognized recurrent breakpoint responsible for rearrangements in the WBS region. Given that most commercial FISH assays include probes flanking this novel breakpoint, further testing with array CGH should be performed in patients with WBS and negative FISH results.

WWOX, the WW domain-containing oxidoreductase gene at chromosome region 16q23.3–q24.1, spanning chromosomal fragile site FRA16D, encodes the 46 kDa Wwox protein, a tumor suppressor that is lost or reduced in expression in a wide variety of cancers, including breast, prostate, ovarian, and lung. The function of Wwox as a tumor suppressor implies that it serves a function in the prevention of carcinogenesis. Indeed, in vitro studies show that Wwox protein interacts with many binding partners to regulate cellular apoptosis, proliferation, and/or maturation. It has been reported that newborn Wwox knockout mice exhibit nascent osteosarcomas while Wwox+/− mice exhibit increased incidence of spontaneous and induced tumors. Furthermore, absence or reduction of Wwox expression in mouse xenograft models results in increased tumorigenesis, which can be rescued by Wwox re-expression, though there is not universal agreement among investigators regarding the role of Wwox loss in these experimental models. Despite this proposed tumor suppressor function, the overlap of the human WWOX locus with FRA16D sensitizes the gene to protein-inactivating deletions caused by replication stress. The high frequency of deletions within the WWOX locus in cancers of various types, without the hallmark protein inactivation-associated mutations of “classical” tumor suppressors, has led to the proposal that WWOX deletions in cancers are passenger events that occur in early cancer progenitor cells due to fragility of the genetic locus, rather than driver events which provide the cancer cell a selective advantage. Recently, a proposed epigenetic cause of chromosomal fragility has suggested a novel mechanism for early fragile site instability and has implications regarding the involvement of tumor suppressor genes at chromosomal fragile sites in cancer. In this review, we provide an overview of the evidence for WWOX as a tumor suppressor gene and put this into the context of fragility associated with the FRA16D locus.

DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice.

Expression of a proof-reading deficient form of mitochondrial DNA (mtDNA) polymerase gamma, POLG, causes early death accompanied by features of premature ageing in mouse. However, the mechanism of cellular senescence remains unresolved. In addition to high levels of point mutations of mtDNA, the POLG mutator mouse harbours linear mtDNAs. Using one- and two-dimensional agarose gel electrophoresis, we show that the linear mtDNAs derive from replication intermediates and are indicative of replication pausing and chromosomal breakage at the accompanying fragile sites. Replication fork arrest is not random but occurs at specific sites close to two cis-elements known as O(H) and O(L). Pausing at these sites may be enhanced in the case of exonuclease-deficient POLG owing to delayed resumption of DNA replication, or replisome instability. In either case, the mtDNA replication cycle is perturbed and this might explain the progeroid features of the POLG mutator mouse.

Homozygous deletions are rare in cancers and often target tumour suppressor genes. Here, we build a compendium of 2218 primary tumours across 12 human cancer types and systematically screen for homozygous deletions, aiming to identify rare tumour suppressors. Our analysis defines 96 genomic regions recurrently targeted by homozygous deletions. These recurrent homozygous deletions occur either over tumour suppressors or over fragile sites, regions of increased genomic instability. We construct a statistical model that separates fragile sites from regions showing signatures of positive selection for homozygous deletions and identify candidate tumour suppressors within those regions. We find 16 established tumour suppressors and propose 27 candidate tumour suppressors. Several of these genes (including MGMT, RAD17, and USP44) show prior evidence of a tumour suppressive function. Other candidate tumour suppressors, such as MAFTRR, KIAA1551, and IGF2BP2, are novel. Our study demonstrates how rare tumour suppressors can be identified through copy number meta-analysis.

Genome instability and cell cycle dysregulation are commonly associated with cancer. DNA replication stress driven by oncogene activation during tumorigenesis is now well established as a source of genome instability. Replication stress generates DNA damage not only during S phase, but also in the subsequent mitosis, where it impacts adversely on chromosome segregation. Some regions of the genome seem particularly sensitive to replication stress-induced instability; most notably, chromosome fragile sites. In this article, we review some of the important issues that have emerged in recent years concerning DNA replication stress and fragile site expression, as well as how chromosome instability is minimized by a family of ring-shaped protein complexes known as SMC proteins. Understanding how replication stress impacts on S phase and mitosis in cancer should provide opportunities for the development of novel and tumour-specific treatments.

A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all individuals) or rare (RFSs; present in only a few individuals). Interest in fragile sites has remained high since their discovery in 1965, because of their association with human disease. CFSs are recognized as drivers of oncogene activation and genome instability in cancer cells, while some RFSs are associated with neurodegenerative diseases. This review summaries our current understanding of the nature and causes of fragile site "expression", including the recently characterized phenomenon of telomere fragility. In particular, we focus on a description of the methodologies and technologies for detection and analysis of chromosome fragile sites.

Ultrafine anaphase bridges (UFBs) are thin DNA threads linking the separating sister chromatids in the anaphase of mitosis. UFBs are thought to form when topological DNA entanglements between two chromatids are not resolved prior to anaphase onset. In contrast to other markers of defective chromosome segregation, UFBs cannot be detected by direct staining of the DNA, but instead can be detected using immunofluorescence-based approaches. Due to the fact that they are short-lived and fragile in nature, UFBs can be challenging to detect. In this chapter, we describe methods that have been optimized for successful detection of UFBs. We also provide guidelines for the optimization of UFBs detection depending on the antibody and the cell line to be used.

Maintaining genome integrity is crucial for normal cellular functions. DNA double-strand breaks (DSBs), when unrepaired, can potentiate chromosomal translocations. t(14;18) translocation involving BCL2 gene on chromosome 18 and IgH loci at chromosome 14, could lead to follicular lymphoma. Molecular basis for fragility of translocation breakpoint regions is an active area of investigation. Previously, formation of non-B DNA structures like G-quadruplex, triplex, B/A transition were investigated at peak I of BCL2 major breakpoint region (MBR); however, it is less understood at peak III. In vitro gel shift assays show faster mobility for MBR peak III sequences, unlike controls. CD studies of peak III sequences reveal a spectral pattern different from B-DNA. Although complementary C-rich stretches exhibit single-strandedness, corresponding guanine-rich sequences do not show DMS protection, ruling out G-quadruplex and triplex DNA. Extrachromosomal assay indicates that peak III halts transcription, unlike its mutated version. Taken together, multiple lines of evidence suggest formation of potential cruciform DNA structure at MBR peak III, which was also supported by in silico studies. Thus, our study reveals formation of non-B DNA structure which could be a basis for fragility at BCL2 breakpoint regions, eventually leading to chromosomal translocations.

Ever since initial suggestions that instability at common fragile sites (CFSs) could be responsible for chromosome rearrangements in cancers, CFSs and associated genes have been the subject of numerous studies, leading to questions and controversies about their role and importance in cancer. It is now clear that CFSs are not frequently involved in translocations or other cancer-associated recurrent gross chromosome rearrangements. However, recent studies have provided new insights into the mechanisms of CFS instability, their effect on genome instability, and their role in generating focal copy number alterations that affect the genomic landscape of many cancers.

Many different processes have an impact on the shape of plant karyotype. Recently, cytogenetic examination of Lolium species has revealed the occurrence of spontaneous fragile sites (FSs) associated with 35S rDNA regions. The FSs are defined as the chromosomal regions that are sensitive to forming gaps or breaks on chromosomes. The shape of karyotype can also be determined by interstitial telomeric sequences (ITSs), what was recognized for the first time in this paper in chromosomes of Festuca pratensis × Lolium perenne hybrids. Both FSs and ITSs can contribute to genome instabilities and chromosome rearrangements. To evaluate whether these cytogenetic phenomena have an impact on karyotype reshuffling observed in Festuca × Lolium hybrids, we examined F1 F. pratensis × L. perenne plants and generated F2-F9 progeny by fluorescent in situ hybridization (FISH) using rDNA sequences, telomere and centromere probes, as well as by genomic in situ hybridization (GISH). Analyses using a combination of FISH and GISH revealed that intergenomic rearrangements did not correspond to FSs but overlapped with ITSs for several analyzed genotypes. It suggests that internal telomeric repeats can affect the shape of F. pratensis × L. perenne karyotypes. However, other factors that are involved in rearrangements and have a more crucial impact could exist, but they are still unknown. Electronic supplementary material The online version of this article (10.1007/s00709-017-1161-5) contains supplementary material, which is available to authorized users.

DNA replication stress is one of the early drivers enabling the ongoing acquisition of genetic changes arising during tumorigenesis. As such, it is a feature of most pre-malignant and malignant cells. In this review article, we focus on the early events initiating DNA replication stress and the preferential sensitivity of common fragile sites (CFSs) to this stress. CFSs are specific genomic regions within the normal chromosomal structure, which appear as gaps and breaks in the metaphase chromosomes of cells grown under mild replication stress conditions. The main characteristics predisposing CFSs to instability include late replication timing, delayed replication completion, failure to activate additional origins, origin paucity along large genomic regions, collision between replication and transcription complexes along large genes, and the presence of AT-dinucleotide rich sequences. The contribution of these features to instability at CFSs during early cancer development is discussed.