The fragile sites are defined as specific segments of genes that are particularly susceptible to breakage under conditions of accelerated replication stress or certain external influences. It has been demonstrated that fragile sites can influence the progression of various tumors. However, the majority of existing studies have focused on the functions of well-characterized common fragile sites, such as FHIT, WWOX, and PARK2, in different oncogenic processes, with insufficient attention directed towards other fragile sites. This article presents an analysis of recent investigations into the fragile sites, fragile site-associated tumor suppressor (FATS) and fragile X mental retardation 1 (FMR1), across various tumor types. The article discusses the mechanisms and signaling pathways regulated by these sites in a range of cancers, as well as their clinical implications for tumor treatment. The review highlights the significance of the fragile sites FATS and FMR1 in various cancers and their clinical relevance.

Recent developments in low-input genomics techniques have greatly advanced the analysis of the order in which DNA is replicated in the genome - that is, replication timing (RT) - and its interrelationships with other processes. RT correlates or anticorrelates with genomic-specific parameters such as gene expression, chromatin accessibility, histone modifications, and the 3D structure of the genome, but the significance of how they influence each other and how they relate to biological processes remains unclear. In this review I discuss the results of recent analyses of RT, the time at which it is remodeled and consolidated during embryogenesis, how it influences development and differentiation, and the regulatory mechanisms and factors involved.

Genomes are blueprints of life essential for an organism's survival, propagation, and evolutionary adaptation. Eukaryotic genomes comprise of DNA, core histones, and several other nonhistone proteins, packaged into chromatin in the tiny confines of nucleus. Chromatin structural organization restricts transcription factors to access DNA, permitting binding only after specific chromatin remodeling events. The fundamental processes in living cells, including transcription, replication, repair, and recombination, are thus regulated by chromatin structure through ATP-dependent remodeling, histone variant incorporation, and various covalent histone modifications including phosphorylation, acetylation, and ubiquitination. These modifications, particularly involving histone variant H2AX, furthermore play crucial roles in DNA damage responses by enabling repair protein's access to damaged DNA. Chromatin also stabilizes the genome by regulating DNA repair mechanisms while suppressing damage from endogenous and exogenous sources. Environmental factors such as ionizing radiations induce DNA damage, and if repair is compromised, can lead to chromosomal abnormalities and gene amplifications as observed in several tumor types. Consequently, chromatin architecture controls the genome fidelity and activity: it orchestrates correct gene expression, genomic integrity, DNA repair, transcription, replication, and recombination. This review considers connecting chromatin organization to functional outcomes impacting transcription, DNA repair and genomic integrity as an emerging grand challenge for predictive molecular cell biology.

Helicobacter pylori (H. pylori) infection is a significant risk factor for gastric cancer (GC) development. A growing body of evidence suggests a causal link between infection with H. pylori and increased DNA breakage in the host cells. While several mechanisms have been proposed for this damage, their relative impact on the overall bacterial genotoxicity is unknown. Moreover, the link between the formation of DNA damage following infection and the emergence of cancerous structural variants (SV) in the genome of infected cells remained unexplored.

We constructed a high-resolution map of genomic H. pylori-induced recurrent break sites using the END-seq method on AGS human gastric cells before and after infection. We next applied END-seq to cycling and arrested cells to identify the role of DNA replication on break formation. Recurrent H. pylori-mediated break sites were further characterized by analyzing published RNA-seq, DRIP-seq, and GRO-seq data at these sites. γH2AX staining and comet assay were used for DNA breakage quantification. Liquid chromatography-mass spectrometry (LC-MS) assay was used to quantify cellular concentrations of dNTPs.

Our data indicated that sites of recurrent H. pylori-mediated DNA breaks are ubiquitous across cell types, localized at replication-related fragile sites, and their breakage is dependent on replication. Consistent with that, we found that H. pylori inflicts nucleotide depletion, and that rescuing the cellular nucleotide pool largely reduced H. pylori-induced DNA breaks. Intriguingly, we found that this genotoxic mechanism operates independently of H. pylori cag pathogenicity island (CagPAI) that encodes for the bacterial type 4 secretion system (T4SS), and its virulence factor, CagA, which was previously implicated in increasing DNA damage by downregulating the DNA damage response. Finally, we show that sites of recurrent H. pylori-mediated breaks coincide with chromosomal deletions observed in patients with intestinal-type GC and that this link potentially elucidates the persistent transcriptional alterations observed in cancer driver genes.

Our findings indicate that dNTP depletion by H. pylori is a key component of its genotoxicity and suggest a link between H. pylori genotoxicity and its oncogenic potential.

Genome maintenance is essential for the integrity of the genetic blueprint, of which only a small fraction is transcribed in higher eukaryotes. DNA lesions occurring in the transcribed genome trigger transcription pausing and transcription-coupled DNA repair. There are two major transcription-coupled DNA repair pathways. The transcription-coupled nucleotide excision repair (TC-NER) pathway has been well studied for decades, while the transcription-coupled homologous recombination repair (TC-HR) pathway has recently gained attention. Importantly, recent studies have uncovered crucial roles of RNA transcripts in TC-HR, opening exciting directions for future research. Transcription also plays pivotal roles in regulating the stability of highly specialized genomic structures such as telomeres, centromeres, and fragile sites. Despite their positive function in genome maintenance, transcription and RNA transcripts can also be the sources of genomic instability, especially when colliding with DNA replication and forming unscheduled pathological RNA:DNA hybrids (R-loops), respectively. Pathological R-loops can result from transcriptional stress, which may be induced by transcription dysregulation. Future investigation into the interplay between transcription and DNA repair will reveal novel molecular bases for genome maintenance and transcriptional stress-associated genomic instability, providing therapeutic targets for human disease intervention.

Genomic structural variants (SVs) greatly impact human health, but much is unknown about the mechanisms that generate the largest class of nonrecurrent alterations. Common fragile sites (CFSs) are unstable loci that provide a model for SV formation, especially large deletions, under replication stress. We study SV junction formation as it occurs in human cell lines by applying error-minimized capture sequencing to CFS DNA harvested after low-dose aphidicolin treatment. SV junctions form throughout CFS genes at a 5-fold higher rate after cells pass from G2 into M-phase. Neither SV formation nor CFS expression depend on mitotic DNA synthesis (MiDAS), an error-prone form of replication active at CFSs. Instead, analysis of tens of thousands of de novo SV junctions combined with DNA repair pathway inhibition reveal a primary role for DNA polymerase theta (POLQ)-mediated end-joining (TMEJ). We propose an important role for mitotic TMEJ in nonrecurrent SV formation genome wide.

A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation.

Oncogenic mutations (such as in KRAS) can dysregulate transcription and replication, leading to transcription-replication conflicts (TRCs). Here, we demonstrate that TRCs are enriched in human pancreatic ductal adenocarcinoma (PDAC) compared to other common solid tumors or normal cells. Several orthogonal approaches demonstrated that TRCs are oncogene dependent. A small interfering RNA (siRNA) screen identified several factors in the base-excision repair (BER) pathway as main regulators of TRCs in PDAC cells. Inhibitors of BER pathway (methoxyamine and CRT) enhanced TRCs. Mechanistically, BER pathway inhibition severely altered RNA polymerase II (RNAPII) and R-loop dynamics at nascent DNA, causing RNAPII trapping and contributing to enhanced TRCs. The ensuing DNA damage activated the ATR-Chk1 pathway. Co-treatment with ATR inhibitor (VX970) and BER inhibitor (methoxyamine) at clinically relevant doses synergistically enhanced DNA damage and reduced cell proliferation in PDAC cells. The study provides mechanistic insights into the regulation of TRCs in PDAC by the BER pathway, which has biologic and therapeutic implications.

Chromothripsis describes the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei. Although micronuclei accumulate DNA double-strand breaks and replication defects throughout interphase, how chromosomes undergo shattering remains unresolved. Using CRISPR-Cas9 screens, we identify a non-canonical role of the Fanconi anemia (FA) pathway as a driver of chromothripsis. Inactivation of the FA pathway suppresses chromosome shattering during mitosis without impacting interphase-associated defects within micronuclei. Mono-ubiquitination of FANCI-FANCD2 by the FA core complex promotes its mitotic engagement with under-replicated micronuclear chromosomes. The structure-selective SLX4-XPF-ERCC1 endonuclease subsequently induces large-scale nucleolytic cleavage of persistent DNA replication intermediates, which stimulates POLD3-dependent mitotic DNA synthesis to prime shattered fragments for reassembly in the ensuing cell cycle. Notably, FA-pathway-induced chromothripsis generates complex genomic rearrangements and extrachromosomal DNA that confer acquired resistance to anti-cancer therapies. Our findings demonstrate how pathological activation of a central DNA repair mechanism paradoxically triggers cancer genome evolution through chromothripsis.

Eukaryotic chromosomal replication occurs in a segmented, temporal manner wherein open euchromatin and compact heterochromatin replicate during early and late S-phase respectively. Using single molecule DNA fiber analyses coupled with cell synchronization, we find that newly synthesized strands remain stable at perturbed forks in early S-phase. Unexpectedly, stalled forks are susceptible to nucleolytic digestion during late replication resulting in defective fork restart. This inherent vulnerability to nascent strand degradation is dependent on fork reversal enzymes and resection nucleases MRE11, DNA2 and EXO1. Inducing chromatin compaction elicits digestion of nascent DNA in response to fork stalling due to reduced association of RAD51 with nascent DNA. Furthermore, RAD51 occupancy at stalled forks in late S-phase is diminished indicating that densely packed chromatin limits RAD51 accessibility to mediate replication fork protection. Genetic analyses reveal that susceptibility of late replicating forks to nascent DNA digestion is dependent on EXO1 via DNA mismatch repair (MMR) and that the BRCA2-mediated replication fork protection blocks MMR from degrading nascent DNA. Overall, our findings illustrate differential regulation of fork protection between early and late replication and demonstrate nascent strand degradation as a critical determinant of heterochromatin instability in response to replication stress.

Antibodies that gain specificity by a large insert encoding for an extra domain were described for the first time in 2016. In malaria-exposed individuals, an exon deriving from the leukocyte-associated immunoglobulin-like 1 (LAIR1) gene integrated via a copy-and-paste insertion into the immunoglobulin heavy chain encoding region. A few years later, a second example was identified, namely a dual exon integration from the leukocyte immunoglobulin-like receptor B1 (LILRB1) gene that is located in close proximity to LAIR1. A dedicated high-throughput characterization of chimeric immunoglobulin heavy chain transcripts unraveled, that insertions from distant genomic regions (including mitochondrial DNA) can contribute to human antibody diversity. This review describes the modalities of insert-containing antibodies. The role of known DNA mobility aspects, such as genomic translocation, gene conversion, and DNA fragility, is discussed in the context of insert-antibody generation. Finally, the review covers why insert antibodies were omitted from the past repertoire analyses and how insert antibodies can contribute to protective immunity or an autoreactive response.

C10orf90, a tumor suppressor, can inhibit the occurrence and development of tumors. Therefore, we investigated the gene function of C10orf90 in various tumors using multiple pan-cancer datasets. Pan-cancer analysis results reveal that the expression levels of C10orf90 vary across different tumors and hold significant value in the clinical diagnosis and prognosis of patients with various tumors. In some cancers, the expression level of C10orf90 is correlated with CNV, DNA methylation, immune subtypes, immune cell infiltration, and drug sensitivity in the tumors. In particular, in COAD, the C10orf90 gene is implicated in multiple processes associated with COAD. Cell experiments demonstrate that C10orf90 suppresses the proliferation and migration of colon cancer cells while promoting apoptosis. In summary, C10orf90 plays a role in the onset and progression of various cancers and could potentially serve as an effective diagnostic and prognostic marker for cancer patients. Notably, in COAD, C10orf90 inhibits the proliferation and migration of colon cancer cells, induces apoptosis, and is linked to the advancement of colon cancer.

DNA damage tolerance pathways that allow for the completion of replication following fork arrest are critical in maintaining genome stability during cell division. The main DNA damage tolerance pathways include strand switching, replication fork reversal and translesion synthesis (TLS). The TLS pathway is mediated by specialised DNA polymerases that can accommodate altered DNA structures during DNA synthesis, and are important in allowing replication to proceed after fork arrest, preventing fork collapse that can generate more deleterious double-strand breaks in the genome. TLS may occur directly at the fork, or at gaps remaining behind the fork, in the process of post-replication repair. Inactivating mutations in the human POLH gene encoding the Y-family DNA polymerase Pol η causes the skin cancer-prone genetic disease xeroderma pigmentosum variant (XPV). Pol η also contributes to chemoresistance during cancer treatment by bypassing DNA lesions induced by anti-cancer drugs including cisplatin. We review the current understanding of the canonical role of Pol η in translesion synthesis following replication arrest, as well as a number of emerging non-canonical roles of the protein in other aspects of DNA metabolism.

Pronuclear transfer has been successfully used in human-assisted reproduction to suppress the adverse effects of a defective oocyte cytoplasm or to bypass an idiopathic developmental arrest. However, the effects of the initial parental genome remodelling in a defective cytoplasm on the subsequent development after pronucleus transfer have not been systematically studied. By performing pronuclear transfer in pre-replication and post-replication mouse embryos, we show that the timing of the procedure plays a critical role. Although apparently morphologically normal blastocysts were obtained in both pre- and post-replication pronuclear transfer groups, post-replication pronuclear transfer led to a decrease in developmental competence and profound changes in embryonic gene expression. By inhibiting the replication in the abnormal cytoplasm before pronuclear transfer into a healthy cytoplasm, the developmental potential of embryos could be largely restored. This shows that the conditions under which the first embryonic replication occurs strongly influence developmental potential. Although pronuclear transfer is the method of choice for mitigating the impact of a faulty oocyte cytoplasm on early development, our results show that the timing of this intervention should be restricted to the pre-replication phase.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.

This article describes a range of high-dimensional data visualization strategies that we have explored for their ability to complement machine learning algorithm predictions derived from MultiFlow® assay results. For this exercise, we focused on seven biomarker responses resulting from the exposure of TK6 cells to each of 126 diverse chemicals over a range of concentrations. Obviously, challenges associated with visualizing seven biomarker responses were further complicated whenever there was a desire to represent the entire 126 chemical data set as opposed to results from a single chemical. Scatter plots, spider plots, parallel coordinate plots, hierarchical clustering, principal component analysis, toxicological prioritization index, multidimensional scaling, t-distributed stochastic neighbor embedding, and uniform manifold approximation and projection are each considered in turn. Our report provides a comparative analysis of these techniques. In an era where multiplexed assays and machine learning algorithms are becoming the norm, stakeholders should find some of these visualization strategies useful for efficiently and effectively interpreting their high-dimensional data.

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unravel the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA reveals co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity uncovers that head-to-head interactions between replication and transcription machinery result in 60% DSB contribution to the head-on compared to 40% for co-directional. Taken together we reveal TTR as a fragile class and show how the linear interaction between transcription and replication impacts genome stability.

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

Human RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, the Xenopus laevis homolog of RECQL4 has been implicated in initiating DNA replication in egg extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (knock-out; KO) or expression of a full-length, mutant protein lacking helicase activity (helicase-dead; HD). Interestingly, both the RECQL4 KO and HD cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed increased rates in the RECQL4 KO cells, which might be indicative of decreased origin firing efficiency. Our results are consistent with human RECQL4 having a less critical role in firing of DNA replication origins, than its budding yeast homolog Sld2.

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.

Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies.

Common fragile sites (CFSs) are regions prone to chromosomal rearrangements, thereby contributing to tumorigenesis. Under replication stress (RS), CFSs often harbor under-replicated DNA regions at the onset of mitosis, triggering homology-directed repair known as mitotic DNA synthesis (MiDAS) to complete DNA replication. In this study, we identified an important role of DNA mismatch repair protein MutSβ (MSH2/MSH3) in facilitating MiDAS and maintaining CFS stability. Specifically, we demonstrated that MutSβ is required for the increased mitotic recombination induced by RS or FANCM loss at CFS-derived AT-rich and structure-prone sequences (CFS-ATs). We also found that MSH3 exhibits synthetic lethality with FANCM. Mechanistically, MutSβ is required for homologous recombination (HR) especially when DNA double-strand break (DSB) ends contain secondary structures. We also showed that upon RS, MutSβ is recruited to Flex1, a specific CFS-AT, in a PCNA-dependent but MUS81-independent manner. Furthermore, MutSβ interacts with RAD52 and promotes RAD52 recruitment to Flex1 following MUS81-dependent fork cleavage. RAD52, in turn, recruits XPF/ERCC1 to remove DNA secondary structures at DSB ends, enabling HR/break-induced replication (BIR) at CFS-ATs. We propose that the specific requirement of MutSβ in processing DNA secondary structures at CFS-ATs underlies its crucial role in promoting MiDAS and maintaining CFS integrity.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that both bovine and mouse cleavage stage embryos progress through S-phase in a defined pattern. Late replicating regions are associated with the nuclear lamina from the first cell cycle after fertilization, and contain few active origins, and few but long genes. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to segregation of soma and germ line. Our studies show that the formation of early and late replicating regions is among the first layers of epigenetic regulation established on the mammalian genome after fertilization.

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.

Difficult-to-Replicate Sequences (DiToRS) are natural impediments in the human genome that inhibit DNA replication under endogenous replication. Some of the most widely-studied DiToRS are A+T-rich, high "flexibility regions," including long stretches of perfect [AT/TA] microsatellite repeats that have the potential to collapse into hairpin structures when in single-stranded DNA (ssDNA) form and are sites of recurrent structural variation and double-stranded DNA (dsDNA) breaks. Currently, it is unclear how these flexibility regions impact DNA replication, greatly limiting our fundamental understanding of human genome stability. To investigate replication through flexibility regions, we utilized FRET to characterize the effects of the major ssDNA-binding complex, RPA, on the structure of perfect [AT/TA]25 microsatellite repeats and also re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with A+T-rich DNA template sequences. The results indicate that [AT/TA]25 sequences adopt hairpin structures that are unwound by RPA and pol δ holoenzymes support dNTP incorporation through the [AT/TA]25 sequences as well as an A+T-rich, non-structure forming sequence. Furthermore, the extent of dNTP incorporation is dependent on the sequence of the DNA template and the concentration of dNTPs. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on the concentration of dNTPs, whereas the effects of RPA on the replication of an A+T-rich, non-structure forming sequence are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how flexibility regions contribute to genome instability.

Mitotic DNA synthesis (MiDAS) is an unusual form of DNA replication that occurs during mitosis. Initially, MiDAS was characterized as a process associated with intrinsically unstable loci known as common fragile sites that occurs after cells experience DNA replication stress (RS). However, it is now believed to be a more widespread "salvage" mechanism that is called upon to complete the duplication of any under-replicated genomic region. Emerging data suggest that MiDAS is a DNA repair process potentially involving two or more pathways working in parallel or sequentially. In this review, we introduce the causes of RS, regions of the human genome known to be especially vulnerable to RS, and the strategies used to complete DNA replication outside of S phase. Additionally, because MiDAS is a prominent feature of aneuploid cancer cells, we will discuss how targeting MiDAS might potentially lead to improvements in cancer therapy.

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.

Chromosomal structural variation (SV) encompasses a heterogenous class of genetic variants that exerts strong influences on human health and disease. Despite their importance, many structural variants (SVs) have remained poorly characterized at even a basic level, a discrepancy predicated upon the technical limitations of prior genomic assays. However, recent advances in genomic technology can identify and localize SVs accurately, opening new questions regarding SV risk factors and their impacts in humans. Here, we first define and classify human SVs and their generative mechanisms, highlighting characteristics leveraged by various SV assays. We next examine the first-ever gapless assembly of the human genome and the technical process of assembling it, which required third-generation sequencing technologies to resolve structurally complex loci. The new portions of that "telomere-to-telomere" and subsequent pangenome assemblies highlight aspects of SV biology likely to develop in the near-term. We consider the strengths and limitations of the most promising new SV technologies and when they or longstanding approaches are best suited to meeting salient goals in the study of human SV in population-scale genomics research, clinical, and public health contexts. It is a watershed time in our understanding of human SV when new approaches are expected to fundamentally change genomic applications.

Error-corrected sequencing of genomic targets enriched by probe-based capture has become a standard approach for detecting single-nucleotide variants (SNVs) and small insertion/deletions (indels) present at very low variant allele frequencies. Less attention has been given to comparable strategies for rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from samples with known SV properties, we demonstrate that duplex sequencing (DuplexSeq), which demands confirmation of variants on both strands of a source DNA molecule, eliminates false SV junctions arising from chimeric PCR. DuplexSeq could not address frequent intermolecular ligation artifacts that arise during Y-adapter addition prior to strand denaturation without requiring multiple source molecules. In contrast, tagmentation libraries coupled with data filtering based on strand family size greatly reduced both artifact classes and enabled efficient and specific detection of single-molecule SV junctions. The throughput of SV capture sequencing (svCapture) and base-level accuracy of DuplexSeq provided detailed views of the microhomology profile and limited occurrence of de novo SNVs near the junctions of hundreds of newly created SVs, suggesting end joining as a possible formation mechanism. The open source svCapture pipeline enables rare SV detection as a routine addition to SNVs/indels in properly prepared capture sequencing libraries.

Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.

Genome integrity requires replication to be completed before chromosome segregation. The DNA-replication checkpoint (DRC) contributes to this coordination by inhibiting CDK1, which delays mitotic onset. Under-replication of common fragile sites (CFSs), however, escapes surveillance, resulting in mitotic chromosome breaks. Here we asked whether loose DRC activation induced by modest stresses commonly used to destabilize CFSs could explain this leakage. We found that tightening DRC activation or CDK1 inhibition stabilizes CFSs in human cells. Repli-Seq and molecular combing analyses showed a burst of replication initiations implemented in mid S-phase across a subset of late-replicating sequences, including CFSs, while the bulk genome was unaffected. CFS rescue and extra-initiations required CDC6 and CDT1 availability in S-phase, implying that CDK1 inhibition permits mistimed origin licensing and firing. In addition to delaying mitotic onset, tight DRC activation therefore supports replication completion of late origin-poor domains at risk of under-replication, two complementary roles preserving genome stability.

Our genomes harbor conserved DNA sequences, known as common fragile sites (CFSs), that are difficult to replicate and correspond to regions of genome instability. Following replication stress, CFS loci give rise to breaks or gaps (termed CFS expression) where under-replicated DNA subsequently undergoes mitotic DNA synthesis (MiDAS). We show that loss of the structure-selective endonuclease GEN1 reduces CFS expression, leading to defects in MiDAS, ultrafine anaphase bridge formation, and DNA damage in the ensuing cell cycle due to aberrant chromosome segregation. GEN1 knockout cells also exhibit an elevated frequency of bichromatid constrictions consistent with the presence of unresolved regions of under-replicated DNA. Previously, the role of GEN1 was thought to be restricted to the nucleolytic resolution of recombination intermediates. However, its ability to cleave under-replicated DNA at CFS loci indicates that GEN1 plays a dual role resolving both DNA replication and recombination intermediates before chromosome segregation.

Oncogene activation creates DNA replication stress (RS) in cancer cells, which can generate under-replicated DNA regions (UDRs) that persist until cells enter mitosis. UDRs also have the potential to generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct 'unscheduled' DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. Previous studies have shown that MiDAS proceeds via a POLD3-dependent pathway that shows some features of break-induced replication. Here, we define how human cells utilize both DNA gap filling (REV1 and Pol ζ) and replicative (Pol δ) DNA polymerases to complete genome duplication following a perturbed S-phase. We present evidence for the existence of a polymerase-switch during MiDAS that is required for new DNA synthesis at UDRs. Moreover, we reveal that, upon oncogene activation, cancer cell survival is significantly compromised when REV1 is depleted, suggesting that REV1 inhibition might be a feasible approach for the treatment of some human cancers.

BRCA1 and BRCA2 play a critical role in a variety of molecular processes related to DNA metabolism, including homologous recombination and mediating the replication stress response. Individuals with mutations in the BRCA1 and BRCA2 (BRCA1/2) genes have a significantly higher risk of developing various types of cancers, especially cancers of the breast, ovary, pancreas, and prostate. Currently, the Food and Drug Administration (FDA) has approved four PARP inhibitors (PARPi) to treat cancers with BRCA1/2 mutations. In this review, we will first summarize the clinical outcomes of the four FDA-approved PARPi in treating BRCA1/2 deficient cancers. We will then discuss evidence supporting the hypothesis that the cytotoxic effect of PARPi is likely due to inducing excessive replication stress at the difficult-to-replicate (DTR) genomic regions in BRCA1/2 mutated tumors. Finally, we will discuss the ongoing preclinical and clinical studies on how to combine the PARPi with immuno-oncology drugs to further improve clinical outcomes.

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( ≳ 40 known), ≤ 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

A major driver of cancer chromosomal instability is replication stress, the slowing or stalling of DNA replication. How replication stress and genomic instability are connected is not known. Aphidicolin-induced replication stress induces breakages at common fragile sites, but the exact causes of fragility are debated, and acute genomic consequences of replication stress are not fully explored.

We characterize DNA copy number alterations (CNAs) in single, diploid non-transformed cells, caused by one cell cycle in the presence of either aphidicolin or hydroxyurea. Multiple types of CNAs are generated, associated with different genomic regions and features, and observed copy number landscapes are distinct between aphidicolin and hydroxyurea-induced replication stress. Coupling cell type-specific analysis of CNAs to gene expression and single-cell replication timing analyses pinpointed the causative large genes of the most recurrent chromosome-scale CNAs in aphidicolin. These are clustered on chromosome 7 in RPE1 epithelial cells but chromosome 1 in BJ fibroblasts. Chromosome arm level CNAs also generate acentric lagging chromatin and micronuclei containing these chromosomes.

Chromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution. Different inducers of replication stress lead to distinctive CNA landscapes providing the opportunity to derive copy number signatures of specific replication stress mechanisms. Single-cell CNA analysis thus reveals the impact of replication stress on the genome, providing insights into the molecular mechanisms which fuel chromosomal instability in cancer.

The acquisition of DNA damage is an early driving event in tumorigenesis. Premalignant lesions show activated DNA damage responses and inactivation of DNA damage checkpoints promotes malignant transformation. However, DNA damage is also a targetable vulnerability in cancer cells. This requires a detailed understanding of the cellular and molecular mechanisms governing DNA integrity. Here, we review current work on DNA damage in tumorigenesis. We discuss DNA double strand break repair, how repair pathways contribute to tumorigenesis, and how double strand breaks are linked to the tumor microenvironment. Next, we discuss the role of oncogenes in promoting DNA damage through replication stress. Finally, we discuss our current understanding on DNA damage in micronuclei and discuss therapies targeting these DNA damage pathways.

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.

A variety of endogenous and exogenous insults are capable of impeding replication fork progression, leading to replication stress. Several SNF2 fork remodelers have been shown to play critical roles in resolving this replication stress, utilizing different pathways dependent upon the nature of the DNA lesion, location on the DNA, and the stage of the cell cycle, to complete DNA replication in a manner preserving genetic integrity. Under certain conditions, however, the attempted repair may lead to additional genetic instability. Cockayne syndrome group B (CSB) protein, a SNF2 chromatin remodeler best known for its role in transcription-coupled nucleotide excision repair, has recently been shown to catalyze fork reversal, a pathway that can provide stability of stalled forks and allow resumption of DNA synthesis without chromosome breakage. Prolonged stalling of replication forks may collapse to give rise to DNA double-strand breaks, which are preferentially repaired by homology-directed recombination. CSB plays a role in repairing collapsed forks by promoting break-induced replication in S phase and early mitosis. In this review, we discuss roles of CSB in regulating the sources of replication stress, replication stress response, as well as the implications of CSB for cancer therapy.

Failure of cells to process toxic double-strand breaks (DSBs) constitutes a major intrinsic source of genome instability, a hallmark of cancer. In contrast with interphase of the cell cycle, canonical repair pathways in response to DSBs are inactivated in mitosis. Although cell cycle checkpoints prevent transmission of DNA lesions into mitosis under physiological condition, cancer cells frequently display mitotic DNA lesions. In this review, we aim to provide an overview of how mitotic cells process lesions that escape checkpoint surveillance. We outline mechanisms that regulate the mitotic DNA damage response and the different types of lesions that are carried over to mitosis, with a focus on joint DNA molecules arising from under-replication and persistent recombination intermediates, as well as DNA catenanes. Additionally, we discuss the processing pathways that resolve each of these lesions in mitosis. Finally, we address the acute and long-term consequences of unresolved mitotic lesions on cellular fate and genome stability.