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STAR Protocols Mar 2023Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we...
Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we describe a protocol to identify at genome wide and at high resolution the genomic sites where MiDAS occurs in cells exposed to aphidicolin. We use EdU incorporation to label nascent DNA in mitotic cells, followed by isolation of the EdU-labeled DNA and next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Groelly et al. (2022) and Macheret et al. (2020)..
Topics: DNA Replication; DNA; Cells, Cultured; Mitosis; DNA Repair
PubMed: 36598851
DOI: 10.1016/j.xpro.2022.101970 -
DNA Repair Feb 2023New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary...
New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary neoplasms (SPNs) as a late consequence of genotoxic cancer therapies with the highest risk among former childhood cancer patients. Since risk factors and predictive biomarkers for therapy-associated SPN remain unknown, we examined the sensitivity to mild replication stress as a driver of genomic instability and carcinogenesis in fibroblasts from 23 long-term survivors of a pediatric first primary neoplasm (FPN), 22 patients with the same FPN and a subsequent SPN, and 22 controls with no neoplasm (NN) using the cytokinesis-block micronucleus (CBMN) assay. Mild replication stress was induced with the DNA-polymerase inhibitor aphidicolin (APH). Fibroblasts from patients with the DNA repair deficiency syndromes Bloom, Seckel, and Fanconi anemia served as positive controls and for validation of the CBMN assay supplemented by analysis of chromosomal aberrations, DNA repair foci (γH2AX/53BP1), and cell cycle regulation. APH treatment resulted in G2/M arrest and underestimation of cytogenetic damage beyond G2, which could be overcome by inhibition of Chk1. Basal micronuclei were significantly increased in DNA repair deficiency syndromes but comparable between NN, FPN, and SPN donors. After APH-induced replication stress, the average yield of micronuclei was significantly elevated in SPN donors compared to FPN (p = 0.013) as well as NN (p = 0.03) donors but substantially lower than for DNA repair deficiency syndromes. Our findings suggest that mild impairment of the response to replication stress induced by genotoxic impacts of DNA-damaging cancer therapies promotes genomic instability in a subset of long-term cancer survivors and may drive the development of an SPN. Our study provides a basis for detailed mechanistic studies as well as predictive bioassays for clinical surveillance, to identify cancer patients at high risk for SPNs at first diagnosis.
Topics: Humans; Child; Cancer Survivors; Neoplasms, Second Primary; Apoptosis; Cell Line, Tumor; G2 Phase Cell Cycle Checkpoints; Chromosomal Instability; Genomic Instability; Micronucleus Tests; DNA Damage; DNA; Fibroblasts
PubMed: 36549044
DOI: 10.1016/j.dnarep.2022.103435 -
Frontiers in Genetics 2022Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent...
Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced by mild level of DNA replication stress, such as DNA polymerase inhibition or nucleotide pool disturbance. The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300 kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.
PubMed: 36506300
DOI: 10.3389/fgene.2022.907547 -
PLoS Genetics Dec 2022DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts...
DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids. RAD18 is an E3 ubiquitin ligase for PCNA that is involved in coordinating DNA damage tolerance pathways to preserve genome stability during replication. In this study, we show that RAD18 deficient cells have higher levels of transcription-replication conflicts and accumulate DNA:RNA hybrids that induce DNA double strand breaks and replication stress. We find that these effects are driven in part by failure to recruit the Fanconi Anemia protein FANCD2 at difficult to replicate and R-loop prone genomic sites. FANCD2 activation caused by splicing inhibition or aphidicolin treatment is critically dependent on RAD18 activity. Thus, we highlight a RAD18-dependent pathway promoting FANCD2-mediated suppression of R-loops and transcription-replication conflicts.
Topics: Humans; DNA Repair; Fanconi Anemia; Ubiquitin-Protein Ligases; Fanconi Anemia Complementation Group D2 Protein; DNA; DNA Damage; DNA Replication; RNA; Genomic Instability; DNA-Binding Proteins
PubMed: 36480547
DOI: 10.1371/journal.pgen.1010309 -
Frontiers in Genetics 2022Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites.... (Review)
Review
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 (mmunodeficiency entromeric instability and acial 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.
PubMed: 36468036
DOI: 10.3389/fgene.2022.985975 -
Nucleic Acids Research Nov 2022In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events...
In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.
Topics: Animals; Mice; Replication Origin; Chromatin; Mouse Embryonic Stem Cells; DNA Replication; Cell Cycle Proteins; Mammals
PubMed: 36453993
DOI: 10.1093/nar/gkac1111 -
Biology Oct 2022In recent years, amniotic fluids have gained attention in cancer research. They have an influential role in protecting embryos against several anomalies. Chick early...
In recent years, amniotic fluids have gained attention in cancer research. They have an influential role in protecting embryos against several anomalies. Chick early amniotic fluid (ceAF)-amniotic fluid isolated from growing chicken-has been used in many other studies, including myocardial infarctions and skin regeneration. In this study, we employed ceAF's promising therapeutic applications against tumorigenesis in both in vitro and in vivo studies. We selected three robust proliferating tumor cell lines: BCaP37, MCF7, and RKO. We found that selective dosage is required to obtain maximum impact to deter tumorigenesis. ceAF not only disrupted the uniform colonies of tumor cell lines via disturbing mitochondrial transmembrane potential, but also arrested many cells at growing G1 state via working agonistically with aphidicolin. The significant inhibition of tumor metastasis by ceAF was indicated by in vivo models. This leads to apoptosis analysis as verified by annexin-V staining stays and immunoblotting of critical proteins as cell cycle meditators and apoptosis regulators. Not only on the protein level, but we also tested ceAF's therapeutic potentials on mRNA levels as indicated by quantitative real-time PCR summarizing the promising role of ceAF in deterring tumor progression. In conclusion, our study reveals the potent role of ceAF against tumorigenesis in breast cancer and colon carcinoma. Further studies will be required to determine the critical components present in ceAF and its purification to narrow down this study.
PubMed: 36358278
DOI: 10.3390/biology11111577 -
Genome Biology Oct 2022A major driver of cancer chromosomal instability is replication stress, the slowing or stalling of DNA replication. How replication stress and genomic instability are...
BACKGROUND
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.
RESULTS
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.
CONCLUSIONS
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.
Topics: Humans; DNA Copy Number Variations; Aphidicolin; Hydroxyurea; Neoplasms; DNA; Chromosomal Instability; Chromosomes; Chromatin
PubMed: 36266663
DOI: 10.1186/s13059-022-02781-0 -
Nature Communications Oct 2022The pathways involved in suppressing DNA replication stress and the associated DNA damage are critical to maintaining genome integrity. The Mre11 complex is unique among...
The pathways involved in suppressing DNA replication stress and the associated DNA damage are critical to maintaining genome integrity. The Mre11 complex is unique among double strand break (DSB) repair proteins for its association with the DNA replication fork. Here we show that Mre11 complex inactivation causes DNA replication stress and changes in the abundance of proteins associated with nascent DNA. One of the most highly enriched proteins at the DNA replication fork upon Mre11 complex inactivation was the ubiquitin like protein ISG15. Mre11 complex deficiency and drug induced replication stress both led to the accumulation of cytoplasmic DNA and the subsequent activation of innate immune signaling via cGAS-STING-Tbk1. This led to ISG15 induction and protein ISGylation, including constituents of the replication fork. ISG15 plays a direct role in preventing replication stress. Deletion of ISG15 was associated with replication fork stalling, tonic ATR activation, genomic aberrations, and sensitivity to aphidicolin. These data reveal a previously unrecognized role for ISG15 in mitigating DNA replication stress and promoting genomic stability.
Topics: Aphidicolin; DNA; DNA Damage; DNA Repair; DNA Replication; Nucleotidyltransferases; Ubiquitins
PubMed: 36216822
DOI: 10.1038/s41467-022-33535-y -
Mutation Research 2022PTEN is a tumor suppressor protein frequently altered in various cancers. PTEN-null cells have a characteristic of rapid proliferation with an unstable genome....
PTEN is a tumor suppressor protein frequently altered in various cancers. PTEN-null cells have a characteristic of rapid proliferation with an unstable genome. Replication stress is one of the causes of the accumulation of genomic instability if not sensed by the cellular signaling. Though PTEN-null cells have shown to be impaired in replication progression and stalled fork recovery, the association between the catalytic function of PTEN regulated by posttranslational modulation and cellular response to replication stress has not been studied explicitly. To understand molecular mechanism, we find that PTEN-null cells display unrestrained replication fork progression with accumulation of damaged DNA after treatment with aphidicolin which can be rescued by ectopic expression of full-length PTEN, as evident from DNA fiber assay. Moreover, the C-terminal phosphorylation (Ser 380, Thr 382/383) of PTEN is essential for its chromatin association and sensing replication stress that, in response, induce cell cycle arrest. Further, we observed that PTEN induces HP1α expression and H3K9me3 foci formation in a C-terminal phosphorylation-dependent manner. However, phosphatase dead PTEN cannot sense replication stress though it can be associated with chromatin. Together, our results suggest that DNA replication perturbation by aphidicolin enables chromatin association of PTEN through C-terminal phosphorylation, induces heterochromatin formation by stabilizing and up-regulating H3K9me3 foci and augments CHK1 activation. Thereby, PTEN prevents DNA replication fork elongation and simultaneously causes G1-S phase cell cycle arrest to limit cell proliferation in stress conditions. Thus PTEN act as stress sensing protein during replication arrest to maintain genomic stability.
Topics: Humans; Phosphorylation; Heterochromatin; Aphidicolin; Chromatin; Chromatin Assembly and Disassembly; Genomic Instability; PTEN Phosphohydrolase
PubMed: 36155262
DOI: 10.1016/j.mrfmmm.2022.111800