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.

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis.

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.

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.

FANCD2 Facilitates Replication through Common Fragile Sites.

Common fragile sites (CFSs) are genomic regions that are unstable under conditions of replicative stress. Although the characteristics of CFSs that render them vulnerable to stress are associated mainly with replication, the cellular pathways that protect CFSs during replication remain unclear. Here, we identify and describe a role for FANCD2 as a trans-acting facilitator of CFS replication, in the absence of exogenous replicative stress. In the absence of FANCD2, replication forks stall within the AT-rich fragility core of CFS, leading to dormant origin activation. Furthermore, FANCD2 deficiency is associated with DNA:RNA hybrid formation at CFS-FRA16D, and inhibition of DNA:RNA hybrid formation suppresses replication perturbation. In addition, we also found that FANCD2 reduces the number of potential sites of replication initiation. Our data demonstrate that FANCD2 protein is required to ensure efficient CFS replication and provide mechanistic insight into how FANCD2 regulates CFS stability.

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.

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.

DNA replication stress and its impact on chromosome segregation and tumorigenesis.

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.

From R-Loops to G-Quadruplexes: Emerging New Threats for the Replication Fork.

Replicating the entire genome is one of the most complex tasks for all organisms. Research carried out in the last few years has provided us with a clearer picture on how cells preserve genomic information from the numerous insults that may endanger its stability. Different DNA repair pathways, coping with exogenous or endogenous threat, have been dissected at the molecular level. More recently, there has been an increasing interest towards intrinsic obstacles to genome replication, paving the way to a novel view on genomic stability. Indeed, in some cases, the movement of the replication fork can be hindered by the presence of stable DNA: RNA hybrids (R-loops), the folding of G-rich sequences into G-quadruplex structures (G4s) or repetitive elements present at Common Fragile Sites (CFS). Although differing in their nature and in the way they affect the replication fork, all of these obstacles are a source of replication stress. Replication stress is one of the main hallmarks of cancer and its prevention is becoming increasingly important as a target for future chemotherapeutics. Here we will try to summarize how these three obstacles are generated and how the cells handle replication stress upon their encounter. Finally, we will consider their role in cancer and their exploitation in current chemotherapeutic approaches.

Mechanisms shaping the mutational landscape of the FRA3B/FHIT-deficient cancer genome.

Genome instability is an enabling characteristic of cancer that facilitates the acquisition of oncogenic mutations that drive tumorigenesis. Underlying much of the instability in cancer is DNA replication stress, which causes both chromosome structural changes and single base-pair mutations. Common fragile sites are some of the earliest and most frequently altered loci in tumors. Notably, the fragile locus, FRA3B, lies within the fragile histidine triad (FHIT) gene, and consequently deletions within FHIT are common in cancer. We review the evidence in support of FHIT as a DNA caretaker and discuss the mechanism by which FHIT promotes genome stability. FHIT increases thymidine kinase 1 (TK1) translation to balance the deoxyribonucleotide triphosphates (dNTPs) for efficient DNA replication. Consequently, FHIT-loss causes replication stress, DNA breaks, aneuploidy, copy-number changes (CNCs), small insertions and deletions, and point mutations. Moreover, FHIT-loss-induced replication stress and DNA breaks cooperate with APOBEC3B overexpression to catalyze DNA hypermutation in cancer, as APOBEC family enzymes prefer single-stranded DNA (ssDNA) as substrates and ssDNA is enriched at sites of both replication stress and DNA breaks. Consistent with the frequent loss of FHIT across a broad spectrum of cancer types, FHIT-deficiency is highly associated with the ubiquitous, clock-like mutation signature 5 occurring in all cancer types thus far examined. The ongoing destabilization of the genome caused by FHIT loss underlies recurrent inactivation of tumor suppressors and activation of oncogenes. Considering that more than 50% of cancers are FHIT-deficient, we propose that FRA3B/FHIT fragility shapes the mutational landscape of cancer genomes.

Common fragile site instability in normal cells: Lessons and perspectives.

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.

The FRA14B common fragile site maps to a region prone to somatic and germline rearrangements within the large GPHN gene.

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 role of fork stalling and DNA structures in causing chromosome fragility.

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.

[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.

Dynamic changes in ORC localization and replication fork progression during tissue differentiation.

BACKGROUND: Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. RESULTS: We exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, we found that sites of ORC binding show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. CONCLUSIONS: These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.

Detours to Replication: Functions of Specialized DNA Polymerases during Oncogene-induced Replication Stress.

Incomplete and low-fidelity genome duplication contribute to genomic instability and cancer development. Difficult-to-Replicate Sequences, or DiToRS, are natural impediments in the genome that require specialized DNA polymerases and repair pathways to complete and maintain faithful DNA synthesis. DiToRS include non B-DNA secondary structures formed by repetitive sequences, for example within chromosomal fragile sites and telomeres, which inhibit DNA replication under endogenous stress conditions. Oncogene activation alters DNA replication dynamics and creates oncogenic replication stress, resulting in persistent activation of the DNA damage and replication stress responses, cell cycle arrest, and cell death. The response to oncogenic replication stress is highly complex and must be tightly regulated to prevent mutations and tumorigenesis. In this review, we summarize types of known DiToRS and the experimental evidence supporting replication inhibition, with a focus on the specialized DNA polymerases utilized to cope with these obstacles. In addition, we discuss different causes of oncogenic replication stress and its impact on DiToRS stability. We highlight recent findings regarding the regulation of DNA polymerases during oncogenic replication stress and the implications for cancer development.

Understanding replication fork progression, stability, and chromosome fragility by exploiting the Suppressor of Underreplication protein.

There are many layers of regulation governing DNA replication to ensure that genetic information is accurately transmitted from mother cell to daughter cell. While much of the control occurs at the level of origin selection and firing, less is known about how replication fork progression is controlled throughout the genome. In Drosophila polytene cells, specific regions of the genome become repressed for DNA replication, resulting in underreplication and decreased copy number. Importantly, underreplicated domains share properties with common fragile sites. The Suppressor of Underreplication protein SUUR is essential for this repression. Recent work established that SUUR functions by directly inhibiting replication fork progression, raising several interesting questions as to how replication fork progression and stability can be modulated within targeted regions of the genome. Here we discuss potential mechanisms by which replication fork inhibition can be achieved and the consequences this has on genome stability and copy number control.

WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response.

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.

Around and beyond 53BP1 Nuclear Bodies.

Within the nucleus, sub-nuclear domains define territories where specific functions occur. Nuclear bodies (NBs) are dynamic structures that concentrate nuclear factors and that can be observed microscopically. Recently, NBs containing the p53 binding protein 1 (53BP1), a key component of the DNA damage response, were defined. Interestingly, 53BP1 NBs are visualized during G1 phase, in daughter cells, while DNA damage was generated in mother cells and not properly processed. Unlike most NBs involved in transcriptional processes, replication has proven to be key for 53BP1 NBs, with replication stress leading to the formation of these large chromatin domains in daughter cells. In this review, we expose the composition and organization of 53BP1 NBs and focus on recent findings regarding their regulation and dynamics. We then concentrate on the importance of the replication stress, examine the relation of 53BP1 NBs with DNA damage and discuss their dysfunction.

Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

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.