A Unifying Hypothesis for the Genome Dynamics Proposed to Underlie Neuropsychiatric Phenotypes.

The sheer number of gene variants and the extent of the observed clinical and molecular heterogeneity recorded in neuropsychiatric disorders (NPDs) could be due to the magnified downstream effects initiated by a smaller group of genomic higher-order alterations in response to endogenous or environmental stress. Chromosomal common fragile sites (CFS) are functionally linked with microRNAs, gene copy number variants (CNVs), sub-microscopic deletions and duplications of DNA, rare single-nucleotide variants (SNVs/SNPs), and small insertions/deletions (indels), as well as chromosomal translocations, gene duplications, altered methylation, microRNA and L1 transposon activity, and 3-D chromosomal topology characteristics. These genomic structural features have been linked with various NPDs in mostly isolated reports and have usually only been viewed as areas harboring potential candidate genes of interest. The suggestion to use a higher level entry point (the 'fragilome' and associated features) activated by a central mechanism ('stress') for studying NPD genetics has the potential to unify the existing vast number of different observations in this field. This approach may explain the continuum of gene findings distributed between affected and unaffected individuals, the clustering of NPD phenotypes and overlapping comorbidities, the extensive clinical and molecular heterogeneity, and the association with certain other medical disorders.

Nascent DNA sequencing and its diverse applications in genome integrity research.

Multiple DNA repair pathways and biological responses to DNA damage have evolved to protect cells from various types of lesions to which they are subjected. Although DNA repair systems are mechanistically distinct, all process the damaged region and then insert new bases to fill the gap. In 1969, Robert Painter developed an assay called "unscheduled" DNA synthesis (UDS), which measures DNA repair synthesis as the uptake of radiolabeled DNA precursors distinct from replicative synthesis. Contemporary detection of nascent DNA during repair by next-generation sequencing grants genome-wide information about the nature of lesions that threaten genome integrity. Recently, we developed the SAR-seq (synthesis associated with repair sequencing) method, which provides a high-resolution view of UDS. SAR-seq has been utilized to map programmed DNA repair sites in non-dividing neurons, replication initiation zones, monitor 53BP1 function in countering end-resection, and to identify regions of the genome that fail to complete replication during S phase but utilize repair synthesis during mitosis (MiDAS). As an example of SAR-seq, we present data showing that sites replicated during mitosis correspond to common fragile sites, which have been linked to tumor progression, cellular senescence, and aging.

Integrated analysis of FHIT gene alterations in cancer.

The Fragile Histidine Triad Diadenosine Triphosphatase (FHIT) gene is located in the Common Fragile Site FRA3B and encodes an enzyme that hydrolyzes the dinucleotide Ap3A. Although FHIT loss is one of the most frequent copy number alterations in cancer, its relevance for cancer initiation and progression remains unclear. FHIT is frequently lost in cancers from the digestive tract, which is compatible with being a cancer driver event in these tissues. However, FHIT loss could also be a passenger event due to the inherent fragility of the FRA3B locus. Moreover, the physiological relevance of FHIT enzymatic activity and the levels of Ap3A is largely unclear. We have conducted here a systematic pan-cancer analysis of FHIT status in connection with other mutations and phenotypic alterations, and we have critically discussed our findings in connection with the literature to provide an overall view of FHIT implications in cancer.

Insights into common fragile site instability: DNA replication challenges at DNA repeat sequences.

Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by their association with copy number variants (CNVs) frequently arise in most tumor types. Over the years, it became clear that a combination of different characteristics underlies the enhanced sensitivity of CFSs to replication stress. As of today, there is a strong evidence that the core fragility regions along CFSs overlap with actively transcribed large genes with delayed replication timing upon replication stress. Recently, the mechanistic basis for CFS instability was further extended to regions which span topologically associated domain (TAD) boundaries, generating a fragility signature composed of replication, transcription and genome organization. The presence of difficult-to-replicate AT-rich repeats was one of the early features suggested to characterize a subgroup of CFSs. These long stretches of AT-dinucleotide have the potential to fold into stable secondary structures which may impede replication fork progression, leaving the region under-replicated. Here, we focus on the molecular mechanisms underlying repeat instability at CFSs and on the proteins involved in the resolution of secondary structure impediments arising along repetitive sequence elements which are essential for the maintenance of genome stability.

Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ.

Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS). We previously demonstrated that a MiDAS mechanism also exists in untransformed and primary human cells, which is RAD52 independent but requires FANCD2. However, the properties of this form of MiDAS are not well understood. Here, we report that FANCD2-driven MiDAS in untransformed human cells: 1) requires a prerequisite step of FANCD2 mono-ubiquitination by a subset of Fanconi anemia (FA) proteins, 2) primarily acts to preserve CFS stability but not to prevent chromosome mis-segregation, and 3) depends on HELQ, which potentially functions at an early step. Hence, FANCD2-driven MiDAS in untransformed cells is built to protect CFS stability, whereas RAD52-driven MiDAS in cancer cells is likely adapted to prevent chromosome mis-segregation at the cost of CFS expression. Notably, we also identified a novel form of MiDAS, which surfaces to function when FANCD2 is absent in untransformed cells. Our findings substantiate the complex nature of MiDAS and a link between its deficiencies and the pathogenesis of FA, a human genetic disease.

Replication stress causes delayed mitotic entry and chromosome 12 fragility at the ANKS1B large neuronal gene in human induced pluripotent stem cells.

Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.

Moonlighting functions of the ubiquitin-like protein, Hub1/UBL-5.

The faithful splicing of pre-mRNA is critical for accurate gene expression. Dysregulation of pre-mRNA splicing has been associated with several human diseases including cancer. The ubiquitin-like protein Hub1/UBL5 binds to the substrates non-covalently and promotes pre-mRNA splicing. Additionally, UBL5 promotes the common fragile sites stability and the Fanconi anemia pathway of DNA damage repair. These functions strongly suggests that UBL5 could potentially be implicated in cancer. Therefore, we analyzed the UBL5 expression in TCGA tumor sample datasets and observed the differences between tumor and normal tissues among different tumor subtypes. We have noticed the alteration frequency of UBL5 in TCGA tumor samples. Altogether, this review summarizes the UBL5 functions and discusses its putative role in tumorigenesis.

Fanconi anemia-associated chromosomal radial formation is dependent on POLθ-mediated alternative end joining.

Activation of the Fanconi anemia (FA) pathway after treatment with mitomycin C (MMC) is essential for preventing chromosome translocations termed "radials." When replication forks stall at MMC-induced interstrand crosslinks (ICLs), the FA pathway is activated to orchestrate ICL unhooking and repair of the DNA break intermediates. However, in FA-deficient cells, how ICL-associated breaks are resolved in a manner that leads to radials is unclear. Here, we demonstrate that MMC-induced radials are dependent on DNA polymerase theta (POLθ)-mediated alternative end joining (A-EJ). Specifically, we show that radials observed in FANCD2-/- cells are dependent on POLθ and DNA ligase III and occur independently of classical non-homologous end joining. Furthermore, treatment of FANCD2-/- cells with POLθ inhibitors abolishes radials and leads to the accumulation of breaks co-localizing with common fragile sites. Uniformly, these observations implicate A-EJ in radial formation and provide mechanistic insights into the treatment of FA pathway-deficient cancers with POLθ inhibitors.

RAD51 protects human cells from transcription-replication conflicts.

Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS. Here, we have identified a key role for RAD51 in preventing transcription-replication conflicts (TRCs) from triggering replication fork breakage. The genomic regions most affected by RAD51 deficiency are characterized by being replicated and transcribed in early S-phase and show significant overlap with loci prone to cancer-associated amplification. Consistent with a role for RAD51 in protecting against transcription-replication conflicts, many of the adverse effects of RAD51 depletion are ameliorated by inhibiting early S-phase transcription. We propose a model whereby RAD51 suppresses fork breakage and subsequent inadvertent amplification of genomic loci prone to experiencing TRCs.

New Era of Mapping and Understanding Common Fragile Sites: An Updated Review on Origin of Chromosome Fragility.

Common fragile sites (CFSs) are specific genomic loci prone to forming gaps or breakages upon replication perturbation, which correlate well with chromosomal rearrangement and copy number variation. CFSs have been actively studied due to their important pathophysiological relevance in different diseases such as cancer and neurological disorders. The genetic locations and sequences of CFSs are crucial to understanding the origin of such unstable sites, which require reliable mapping and characterizing approaches. In this review, we will inspect the evolving techniques for CFSs mapping, especially genome-wide mapping and sequencing of CFSs based on current knowledge of CFSs. We will also revisit the well-established hypotheses on the origin of CFSs fragility, incorporating novel findings from the comprehensive analysis of finely mapped CFSs regarding their locations, sequences, and replication/transcription, etc. This review will present the most up-to-date picture of CFSs and, potentially, a new framework for future research of CFSs.

Lost by Transcription: Fork Failures, Elevated Expression, and Clinical Consequences Related to Deletions in Metastatic Colorectal Cancer.

Among the structural variants observed in metastatic colorectal cancer (mCRC), deletions (DELs) show a size preference of ~10 kb-1 Mb and are often found in common fragile sites (CFSs). To gain more insight into the biology behind the occurrence of these specific DELs in mCRC, and their possible association with outcome, we here studied them in detail in metastatic lesions of 429 CRC patients using available whole-genome sequencing and corresponding RNA-seq data. Breakpoints of DELs within CFSs are significantly more often located between two consecutive replication origins compared to DELs outside CFSs. DELs are more frequently located at the midpoint of genes inside CFSs with duplications (DUPs) at the flanks of the genes. The median expression of genes inside CFSs was significantly higher than those of similarly-sized genes outside CFSs. Patients with high numbers of these specific DELs showed a shorter progression-free survival time on platinum-containing therapy. Taken together, we propose that the observed DEL/DUP patterns in expressed genes located in CFSs are consistent with a model of transcription-dependent double-fork failure, and, importantly, that the ability to overcome the resulting stalled replication forks decreases sensitivity to platinum-containing treatment, known to induce stalled replication forks as well. Therefore, we propose that our DEL score can be used as predictive biomarker for decreased sensitivity to platinum-containing treatment, which, upon validation, may augment future therapeutic choices.

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites.

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

A method to sequence genomic sites of mitotic DNA synthesis in mammalian cells.

Under normal conditions, the genome of eukaryotic cells is faithfully replicated during S phase. However, in cells exposed to DNA polymerase inhibitors, some regions of the genome may fail to be replicated prior to mitotic entry. To prevent chromosomal breakage and loss of genomic information, mitotic DNA synthesis (MiDAS) completes replication of the genome prior to the onset of anaphase. We have developed a protocol that allows one to map the genomic regions that are replicated by MiDAS in mammalian cells. The protocol involves incorporation of a thymidine analog in nascent DNA in mitotic cells and then capture and high throughput sequencing of the nascent DNA. With this approach, sites of MiDAS can be identified at high resolution.

Consequences and Resolution of Transcription-Replication Conflicts.

Transcription-replication conflicts occur when the two critical cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance. In this article, we summarize our current knowledge on where and when collisions occur and how these encounters affect the genome and chromatin landscape of cells. Finally, we conclude with the different cellular pathways and multiple mechanisms that cells have put in place at conflict sites to ensure the resolution of conflicts and accurate genome duplication.

Mind the replication gap.

Unlike bacteria, mammalian cells need to complete DNA replication before segregating their chromosomes for the maintenance of genome integrity. Thus, cells have evolved efficient pathways to restore stalled and/or collapsed replication forks during S-phase, and when necessary, also to delay cell cycle progression to ensure replication completion. However, strong evidence shows that cells can proceed to mitosis with incompletely replicated DNA when under mild replication stress (RS) conditions. Consequently, the incompletely replicated genomic gaps form, predominantly at common fragile site regions, where the converging fork-like DNA structures accumulate. These branched structures pose a severe threat to the faithful disjunction of chromosomes as they physically interlink the partially duplicated sister chromatids. In this review, we provide an overview discussing how cells respond and deal with the under-replicated DNA structures that escape from the S/G2 surveillance system. We also focus on recent research of a mitotic break-induced replication pathway (also known as mitotic DNA repair synthesis), which has been proposed to operate during prophase in an attempt to finish DNA synthesis at the under-replicated genomic regions. Finally, we discuss recent data on how mild RS may cause chromosome instability and mutations that accelerate cancer genome evolution.

[The relationship between HPV integration and prognosis of cervical cancer].

Objective: To investigate the relationship between human papillomavirus (HPV) integration and prognosis of cervical cancer patients. Methods: The data of 82 patients with cervical cancer treated in the Radiotherapy Department of Peking Union Medical College Hospital from October 2004 to June 2012 were retrospectively analyzed.The patients were divided into poor prognosis group (recurrence or metastasis after surgery and adjuvant radiotherapy) and good prognosis group based on a propensity score matching strategy.The HPV integration of the two groups were detected by whole exome sequencing to determine whether the integration sites were located in the common fragile sites (CFSs). HPV integration and integration into CFSs were compared between the two groups. Results: Among the enrolled 82 patients, 37 were divided in poor survival group and 45 in good survival group. A total of 90 integration breakpoints were identified, 30 of them occurred in poor prognosis group and 60 occurred in good prognosis group. In the poor prognosis group, HPV integration occurred in 20 patients, 13 of them were inserted in CFSs of 11 patients, and the numbers in good prognosis group were 26, 17, 11, respectively. There were no significantly statistical differences in the number of HPV integration events (P=0.289), HPV integration patients (P=0.735), CFSs integration events (P=0.427), and CFSs integration patients (P=0.591) between the two groups. In poor prognosis group, more CFSs integration events occurred in patients with metastasis than those in patients with only local recurrence (9 vs 2, P=0.003). Conclusions: No significant differences are observed in HPV integration and HPV integration into CFSs between cervical cancer patients with different prognoses. HPV integration into CFSs may be associated with distant metastasis.

Under-Replicated DNA: The Byproduct of Large Genomes?

In this review, we provide an overview of how proliferating eukaryotic cells overcome one of the main threats to genome stability: incomplete genomic DNA replication during S phase. We discuss why it is currently accepted that double fork stalling (DFS) events are unavoidable events in higher eukaryotes with large genomes and which responses have evolved to cope with its main consequence: the presence of under-replicated DNA (UR-DNA) outside S phase. Particular emphasis is placed on the processes that constrain the detrimental effects of UR-DNA. We discuss how mitotic DNA synthesis (MiDAS), mitotic end joining events and 53BP1 nuclear bodies (53BP1-NBs) deal with such specific S phase DNA replication remnants during the subsequent phases of the cell cycle.

Common Fragile Sites Are Characterized by Faulty Condensin Loading after Replication Stress.

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.

Impaired Replication Timing Promotes Tissue-Specific Expression of Common Fragile Sites.

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.