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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 -
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 -
Nature Communications Mar 2023Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show...
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.
Topics: Humans; Reactive Oxygen Species; DNA Replication; S Phase; DNA-Binding Proteins; Hydroxyurea; DNA
PubMed: 36997515
DOI: 10.1038/s41467-023-37341-y -
Molecular Cell Sep 2022Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS)....
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.
Topics: Aphidicolin; BRCA2 Protein; Chromosome Fragile Sites; DNA; DNA Damage; DNA Replication; Genomic Instability; Humans; Mitosis
PubMed: 36002001
DOI: 10.1016/j.molcel.2022.07.011 -
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 -
Cell Reports Dec 2023Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive...
Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive understanding of how PCNA K164 ubiquitination promotes genome stability. To evaluate this, we generated stable cell lines expressing PCNA from the endogenous PCNA locus. Our data reveal that the inability to ubiquitinate K164 causes perturbations in global DNA replication. Persistent replication stress generates under-replicated regions and is exacerbated by the DNA polymerase inhibitor aphidicolin. We show that these phenotypes are due, in part, to impaired Fanconi anemia group D2 protein (FANCD2)-dependent mitotic DNA synthesis (MiDAS) in PCNA cells. FANCD2 mono-ubiquitination is significantly reduced in PCNA mutants, leading to reduced chromatin association and foci formation, both prerequisites for FANCD2-dependent MiDAS. Furthermore, K164 ubiquitination coordinates direct PCNA/FANCD2 colocalization in mitotic nuclei. Here, we show that PCNA K164 ubiquitination maintains human genome stability by promoting FANCD2-dependent MiDAS to prevent the accumulation of under-replicated DNA.
Topics: Humans; DNA; DNA Damage; DNA Repair; DNA Replication; Fanconi Anemia Complementation Group D2 Protein; Genomic Instability; Proliferating Cell Nuclear Antigen; Ubiquitination
PubMed: 38060446
DOI: 10.1016/j.celrep.2023.113523 -
Frontiers in Genetics 2021Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system (CNS). The poor prognosis of GBM due to resistance to therapy has been associated with...
Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system (CNS). The poor prognosis of GBM due to resistance to therapy has been associated with high chromosomal instability (CIN). Replication stress is a major cause of CIN that manifests as chromosome rearrangements, fragility, and breaks, including those cytologically expressed within specific chromosome regions named common fragile sites (CFSs). In this work, we characterized the expression of human CFSs in the glioblastoma U-251 MG cell line upon treatment with the inhibitor of DNA polymerase alpha aphidicolin (APH). We observed 52 gaps/breaks located within previously characterized CFSs. We found 17 to be CFSs in GBM cells upon treatment with APH, showing a frequency equal to at least 1% of the total gaps/breaks. We report that two CFSs localized to regions FRA2E (2p13/p12) and FRA2F (2q22) were only found in U-251 MG cells, but not lymphocytes or fibroblasts, after APH treatment. Notably, these glioblastoma-specific CFSs had a relatively high expression compared to the other CFSs with breakage frequency between ∼7 and 9%. Presence of long genes, incomplete replication, and delayed DNA synthesis during mitosis (MiDAS) after APH treatment suggest that an impaired replication process may contribute to this loci-specific fragility in U-251 MG cells. Altogether, our work offers a characterization of common fragile site expression in glioblastoma U-251 MG cells that may be further exploited for cytogenetic and clinical studies to advance our understanding of this incurable cancer.
PubMed: 35154254
DOI: 10.3389/fgene.2021.810793 -
Frontiers in Genetics 2021The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile... (Review)
Review
The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome.
PubMed: 34567068
DOI: 10.3389/fgene.2021.708860 -
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 -
Nature Communications Nov 2023Keeping replication fork stable is essential for safeguarding genome integrity; hence, its protection is highly regulated. The CTC1-STN1-TEN1 (CST) complex protects...
Keeping replication fork stable is essential for safeguarding genome integrity; hence, its protection is highly regulated. The CTC1-STN1-TEN1 (CST) complex protects stalled forks from aberrant MRE11-mediated nascent strand DNA degradation (NSD). However, the activation mechanism for CST at forks is unknown. Here, we report that STN1 is phosphorylated in its intrinsic disordered region. Loss of STN1 phosphorylation reduces the replication stress-induced STN1 localization to stalled forks, elevates NSD, increases MRE11 access to stalled forks, and decreases RAD51 localization at forks, leading to increased genome instability under perturbed DNA replication condition. STN1 is phosphorylated by both the ATR-CHK1 and the calcium-sensing kinase CaMKK2 in response to hydroxyurea/aphidicolin treatment or elevated cytosolic calcium concentration. Cancer-associated STN1 variants impair STN1 phosphorylation, conferring inability of fork protection. Collectively, our study uncovers that CaMKK2 and ATR-CHK1 target STN1 to enable its fork protective function, and suggests an important role of STN1 phosphorylation in cancer development.
Topics: Humans; Calcium; DNA Replication; Genomic Instability; Hydroxyurea; Neoplasms
PubMed: 38036565
DOI: 10.1038/s41467-023-43685-2