Common fragile sites are loci that preferentially form gaps and breaks on metaphase chromosomes when DNA synthesis is perturbed, particularly after treatment with the DNA polymerase inhibitor, aphidicolin. We and others have identified several cell cycle checkpoint and DNA repair proteins that influence common fragile site stability. However, the initial events underlying fragile site breakage remain poorly understood. We demonstrate here that aphidicolin-induced gaps and breaks at fragile sites are prevented when cells are co-treated with low concentrations of the topoisomerase I inhibitor, camptothecin. This reduction in breakage is accompanied by a reduction in aphidicolin-induced RPA foci, CHK1 and RPA2 phosphorylation, and PCNA monoubiquitination, indicative of reduced levels of single stranded DNA. Furthermore, camptothecin reduces spontaneous fragile site breakage seen in cells lacking ATR, even in the absence of aphidicolin. These data from cultured human cells demonstrate that topoisomerase I activity is required for DNA common fragile site breaks and suggest that polymerase-helicase uncoupling is a key initial event in this process.

Repetitive DNA regions are known as fragile chromosomal sites which present a high flexibility and low stability. Our focus was characterize fragile sites in 5S rDNA regions. The Ancistrus sp. species shows a diploid number of 50 and an indicative Robertsonian fusion at chromosomal pair 1. Two sequences of 5S rDNA were identified: 5S.1 rDNA and 5S.2 rDNA. The first sequence gathers the necessary structures to gene expression and shows a functional secondary structure prediction. Otherwise, the 5S.2 rDNA sequence does not contain the upstream sequences that are required to expression, furthermore its structure prediction reveals a nonfunctional ribosomal RNA. The chromosomal mapping revealed several 5S.1 and 5S.2 rDNA clusters. In addition, the 5S.2 rDNA clusters were found in acrocentric and metacentric chromosomes proximal regions. The pair 1 5S.2 rDNA cluster is co-located with interstitial telomeric sites (ITS). Our results indicate that its clusters are hotspots to chromosomal breaks. During the meiotic prophase bouquet arrangement, double strand breaks (DSBs) at proximal 5S.2 rDNA of acrocentric chromosomes could lead to homologous and non-homologous repair mechanisms as Robertsonian fusions. Still, ITS sites provides chromosomal instability, resulting in telomeric recombination via TRF2 shelterin protein and a series of breakage-fusion-bridge cycles. Our proposal is that 5S rDNA derived sequences, act as chromosomal fragile sites in association with some chromosomal rearrangements of Loricariidae.

Mutations in the BREVIPEDICELLUS (BP) gene of Arabidopsis thaliana condition a pleiotropic phenotype featuring defects in internode elongation, the homeotic conversion of internode to node tissue, and downward pointing flowers and pedicels. We have characterized five mutant alleles of BP, generated by EMS, fast neutrons, x-rays, and aberrant T-DNA insertion events. Curiously, all of these mutagens resulted in large deletions that range from 140 kbp to over 900 kbp just south of the centromere of chromosome 4. The breakpoints of these mutants were identified by employing inverse PCR and DNA sequencing. The south breakpoints of all alleles cluster in BAC T12G13, while the north breakpoint locations are scattered. With the exception of a microhomology at the bp-5 breakpoint, there is no homology in the junction regions, suggesting that double-stranded breaks are repaired via non-homologous end joining. Southwestern blotting demonstrated the presence of nuclear matrix binding sites in the south breakpoint cluster (SBC), which is A/T rich and possesses a variety of repeat sequences. In situ hybridization on pachytene chromosome spreads complemented the molecular analyses and revealed heretofore unrecognized structural variation between the Columbia and Landsberg erecta genomes. Data mining was employed to localize other large deletions around the HY4 locus to the SBC region and to show that chromatin modifications in the region shift from a heterochromatic to euchromatic profile. Comparisons between the BP/HY4 regions of A. lyrata and A. thaliana revealed that several chromosome rearrangement events have occurred during the evolution of these two genomes. Collectively, the features of the region are strikingly similar to the features of characterized metazoan chromosome fragile sites, some of which are associated with karyotype evolution.

Common fragile sites (CFSs) represent large, highly unstable regions of the human genome. CFS sequences are sensitive to perturbation of replication; however, the molecular basis for the instability at CFSs is poorly understood. We hypothesized that a unique epigenetic pattern may underlie the unusual sensitivity of CFSs to replication interference. To examine this hypothesis, we analyzed chromatin modification patterns within the six human CFSs with the highest levels of breakage, and their surrounding non-fragile regions (NCFSs). Chromatin at most of the CFSs analyzed has significantly less histone acetylation than that of their surrounding NCFSs. Trichostatin A and/or 5-azadeoxycytidine treatment reduced chromosome breakage at CFSs. Furthermore, chromatin at the most commonly expressed CFS, the FRA3B, is more resistant to micrococcal nuclease than that of the flanking non-fragile sequences. These results demonstrate that histone hypoacetylation is a characteristic epigenetic pattern of CFSs, and chromatin within CFSs might be relatively more compact than that of the NCFSs, indicating a role for chromatin conformation in genomic instability at CFSs. Moreover, lack of histone acetylation at CFSs may contribute to the defective response to replication stress characteristic of CFSs, leading to the genetic instability characteristic of this regions.

Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council cell strain 5 (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene; interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene (CNTNAP2) in a lymphocytes' CFS, and Duchenne muscular dystrophy gene (DMD) in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability.

Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.

Genome instability is a hallmark of cancer. Common fragile sites (CFSs) are specific regions in the human genome that are sensitive to replication stress and are prone to genomic instability in different cancer types. Here we molecularly cloned a new CFS, FRA11H, in 11q13. The genomic region of FRA11H harbors a hotspot of chromosomal breakpoints found in different types of cancer, indicating that this region is unstable during cancer development. We further found that FRA11H is a hotspot for integrations of Murine Leukemia Virus (MLV)-based vectors, following CD34+ infections in vitro as well as ex-vivo during gene therapy trials. Importantly, we found that the MLV-based vector infection in-vitro leads to replication perturbation, DNA damage and increased CFS expression. This suggests that infection by MLV-based vectors leads to replication-induced genome instability, raising further concerns regarding the use of retroviral vectors in gene therapy trials.

Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics.

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription-replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription-replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

A high level of cytogenetic expression of the rare folate-sensitive fragile site FRA12A is significantly associated with mental retardation. Here, we identify an elongated polymorphic CGG repeat as the molecular basis of FRA12A. This repeat is in the 5' untranslated region of the gene DIP2B, which encodes a protein with a DMAP1-binding domain, which suggests a role in DNA methylation machinery. DIP2B mRNA levels were halved in two subjects with FRA12A with mental retardation in whom the repeat expansion was methylated. In two individuals without mental retardation but with an expanded and methylated repeat, DIP2B expression was reduced to approximately two-thirds of the values observed in controls. Interestingly, a carrier of an unmethylated CGG-repeat expansion showed increased levels of DIP2B mRNA, which suggests that the repeat elongation increases gene expression, as previously described for the fragile X-associated tremor/ataxia syndrome. These data suggest that deficiency of DIP2B, a brain-expressed gene, may mediate the neurocognitive problems associated with FRA12A.

Chromosome inversions are important contributors to standing genetic variation in Drosophila subobscura. Presently, the species is experiencing a rapid replacement of high-latitude by low-latitude inversions associated with global warming. Yet not all low-latitude inversions are correlated with the ongoing warming trend. This is particularly unexpected in the case of O7 because it shows a regular seasonal cycle that peaks in summer and rose with a heatwave. The inconsistent behavior of O7 across components of the ambient temperature suggests that is causally more complex than simply due to temperature alone. In order to understand the dynamics of O7, high-quality genomic data are needed to determine both the breakpoints and the genetic content. To fill this gap, here we generated a PacBio long read-based chromosome-scale genome assembly, from a highly homozygous line made isogenic for an O3 + 4 + 7 chromosome. Then we isolated the complete continuous sequence of O7 by conserved synteny analysis with the available reference genome. Main findings include the following: (i) the assembled O7 inversion stretches 9.936 Mb, containing > 1,000 annotated genes; (ii) O7 had a complex origin, involving multiple breaks associated with non-B DNA-forming motifs, formation of a microinversion, and ectopic repair in trans with the two homologous chromosomes; (iii) the O7 breakpoints carry a pre-inversion record of fragility, including a sequence insertion, and transposition with later inverted duplication of an Attacin immunity gene; and (iv) the O7 inversion relocated the major insulin signaling forkhead box subgroup O (foxo) gene in tight linkage with its antagonistic regulatory partner serine/threonine-protein kinase B (Akt1) and disrupted concerted evolution of the two inverted Attacin duplicates, reattaching them to dFOXO metabolic enhancers. Our findings suggest that O7 exerts antagonistic pleiotropic effects on reproduction and immunity, setting a framework to understand its relationship with climate change. Furthermore, they are relevant for fragility in genome rearrangement evolution and for current views on the contribution of breakage versus repair in shaping inversion-breakpoint junctions.

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.

Autism is a highly heritable complex neurodevelopmental disorder, therefore identifying its genetic basis has been challenging. To date, numerous susceptibility genes and chromosomal abnormalities have been reported in association with autism, but most discoveries either fail to be replicated or account for a small effect. Thus, in most cases the underlying causative genetic mechanisms are not fully understood. In the present work, the Autism Genetic Database (AGD) was developed as a literature-driven, web-based, and easy to access database designed with the aim of creating a comprehensive repository for all the currently reported genes and genomic copy number variations (CNVs) associated with autism in order to further facilitate the assessment of these autism susceptibility genetic factors.

Gene rearrangements such as chromosomal translocations have been shown to contribute to cancer development. Human chromosomal fragile sites are regions of the genome especially prone to breakage, and have been implicated in various chromosome abnormalities found in cancer. However, there has been no comprehensive and quantitative examination of the location of fragile sites in relation to all chromosomal aberrations.