scholarly journals Replication stress induces 53BP1-containing OPT domains in G1 cells

2011 ◽  
Vol 193 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Jeanine A. Harrigan ◽  
Rimma Belotserkovskaya ◽  
Julia Coates ◽  
Daniela S. Dimitrova ◽  
Sophie E. Polo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Immunoprecipitation ◽  
Massively Parallel Sequencing ◽  
Fragile Sites ◽  
Nuclear Bodies ◽  
Sequencing Analysis ◽  
Metaphase Chromosomes ◽  
Common Fragile Sites ◽  
Damage Response ◽  
G1 Cells

Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (γH2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of γH2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.

scholarly journals TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

2010 ◽  
Vol 30 (12) ◽  
pp. 2971-2982 ◽  
Author(s):  
Eladio Abreu ◽  
Elena Aritonovska ◽  
Patrick Reichenbach ◽  
Gaël Cristofari ◽  
Brad Culp ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Immunoprecipitation ◽  
Shelterin Complex ◽  
Damage Response ◽  
Single Stranded Region ◽  
Ob Fold ◽  
Per Se ◽  
Human Telomerase

ABSTRACT Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres—TPP1 and the protein that tethers TPP1 to the complex, TIN2—results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.

scholarly journals Multinucleation Associated DNA Damage causes quiescence despite compromised p53

2020 ◽  
Author(s):  
Madeleine Hart ◽  
Sophie D Adams ◽  
Viji M Draviam
Keyword(s):  
Dna Damage ◽  
Cell Fate ◽  
Cancer Progression ◽  
Cell Tracking ◽  
Nuclear Bodies ◽  
Protein Accumulation ◽  
Tumour Heterogeneity ◽  
Nuclear Atypia ◽  
Significant Difference ◽  
G1 Cells

ABSTRACTNuclear atypia is one of the earliest hallmarks of cancer progression. How distinct forms of nuclear atypia differently impact cell fate is not understood at the molecular level. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of multinucleation or misshapen nuclei and reveal a significant difference between multinucleation and micronucleation, a catastrophic nuclear atypia known to promote genomic rearrangements and tumour heterogeneity. Tracking the fate of newborn cells exhibiting various nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because compromised p53 is seen in over 50% of cancers, we explored how multinucleation blocks proliferation and promotes quiescence. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Importantly, Multinucleation Associated DNA Damage (MADD) associated 53BP1-bodies remain unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. This persistent MADD signalling blocks the onset of DNA replication and is associated with driving proliferative G1 cells into quiescence, revealing a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

scholarly journals 53BP1 mediated recruitment of RASSF1A at ribosomal DNA breaks facilitates local ATM signal amplification

2021 ◽  
Author(s):  
Stavroula Tsaridou ◽  
Georgia Velimezi ◽  
Frances Willenbrock ◽  
Maria Chatzifrangkeskou ◽  
Andreas Panagopoulos ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Viability ◽  
Copy Number ◽  
Adaptor Proteins ◽  
Fragile Sites ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Damage Response ◽  
Rdna Copy ◽  
Rdna Copy Number

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a disproportionally greater impact on the overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites due to repetitive nature, clustering and high transcriptional activity. Recent progress in understanding how the rDNA damage response is organized has highlighted the key role of adaptor proteins in the response. Here we identify that the scaffold and tumor suppressor, RASSF1A is recruited at sites of damage and enriched at rDNA breaks. Employing targeted nucleolar DNA damage, we find that RASSF1A recruitment requires ATM activity and depends on 53BP1. At sites of damage RASSF1A facilitates local ATM signal establishment and rDNA break repair. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Meta-analysis of a lung adenocarcinoma cohort showed that RASSF1A epigenetic silencing leads in rDNA copy number discrepancies. Overall, we present evidence that RASSF1A acts as a DNA repair factor and offer mechanistic insight in how the nucleolar DNA damage response is organized.

scholarly journals Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies

2021 ◽  
Author(s):  
Myriam Scherer ◽  
Clarissa Read ◽  
Gregor Neusser ◽  
Christine Kranz ◽  
Regina Müller ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Viral Infections ◽  
Nuclear Bodies ◽  
Viral Capsids ◽  
Viral Genomes ◽  
Dna Labeling ◽  
Pml Nuclear Bodies ◽  
Damage Response ◽  
Viral Regulatory Proteins

ABSTRACTPML nuclear bodies (PML-NBs) are dynamic interchromosomal macromolecular complexes implicated in epigenetic regulation as well as antiviral defense. During herpesvirus infection, PML-NBs induce epigenetic silencing of viral genomes, however, this defense is antagonized by viral regulatory proteins such as IE1 of human cytomegalovirus (HCMV). Here, we show that PML-NBs undergo a drastic rearrangement into highly enlarged PML cages upon infection with IE1-deficient HCMV. Importantly, our results demonstrate that dual signaling by interferon and DNA damage response is required to elicit giant PML-NBs. DNA labeling revealed that invading HCMV genomes are entrapped inside PML-NBs and remain stably associated with PML cages in a transcriptionally repressed state. Intriguingly, by correlative light and transmission electron microscopy (EM), we observed that PML cages also entrap newly assembled viral capsids demonstrating a second defense layer in cells with incomplete first line response. Further characterization by 3D EM showed that hundreds of viral capsids are tightly packed into several layers of fibrous PML. Overall, our data indicate that giant PML-NBs arise via combined interferon and DNA damage signaling which triggers entrapment of both nucleic acids and proteinaceous components. This represents a multilayered defense strategy to act in a cytoprotective manner and to combat viral infections.

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

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 326 ◽  
Author(s):  
Klizia Maccaroni ◽  
Elisa Balzano ◽  
Federica Mirimao ◽  
Simona Giunta ◽  
Franca Pelliccia
Keyword(s):  
Dna Replication ◽  
Medical Research ◽  
Replication Timing ◽  
Fragile Sites ◽  
Chromosome 1 ◽  
Metaphase Chromosomes ◽  
Specific Expression ◽  
Common Fragile Sites ◽  
Tissue Specific ◽  
Long Genes

Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council cell strain 5 (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene; interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene (CNTNAP2) in a lymphocytes’ CFS, and Duchenne muscular dystrophy gene (DMD) in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.

scholarly journals The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2444
Author(s):  
Tareq Abualfaraj ◽  
Nafiseh Chalabi Hagkarim ◽  
Robert Hollingworth ◽  
Laura Grange ◽  
Satpal Jhujh ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genomic Instability ◽  
Dna Damage Response ◽  
Present Report ◽  
Human Fibroblasts ◽  
Fragile Sites ◽  
Early Region ◽  
Damage Response ◽  
Cellular Dna

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability.

scholarly journals Translation Efficiency and Degradation of ER-Associated mRNAs Modulated by ER-Anchored Poly(A)-Specific Ribonuclease (PARN)

2020 ◽  
Author(s):  
Tian-Li Duan ◽  
Han Jiao ◽  
Guang-Jun He ◽  
Yong-Bin Yan
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Translation Regulation ◽  
Small Subset ◽  
Sequencing Analysis ◽  
Translation Efficiency ◽  
Mouse Tissues ◽  
Damage Response ◽  
Mrna Degradation Rate

Translation is spatiotemporally regulated and ER-associated mRNAs are generally in efficient translation. It is unclear whether the ER-associated mRNAs are deadenylated or degraded on the ER surface in situ or in the cytosol. Here, we showed that ER possessed active deadenylases, particularly the poly(A)-specific ribonuclease (PARN), in common cell lines and mouse tissues. Consistently, purified recombinant PARN exhibited a strong ability to insert into the Langmuir monolayer and liposome. ER-anchored PARN was found to be able to reshape the poly(A) length profile of the ER-associated RNAs by suppressing long poly(A) tails without significantly influencing the cytosolic RNAs. The shortening of long poly(A) tails did not affect global translation efficiency, suggesting that the non-specific action of PARN towards long poly(A) tails was beyond the scope of translation regulation on the ER surface. Transcriptome sequencing analysis indicated that the ER-anchored PARN trigged the degradation of a small subset of ER-enriched transcripts. The ER-anchored PARN modulated the translation of its targets by redistributing ribosomes to heavy polysomes, suggesting that PARN may play a role in dynamic ribosome reallocation. During DNA damage response, MK2 phosphorylated PARN-Ser557 to modulate PARN translocation from the ER to cytosol. By promoting the decay of ER-associated MDM2 transcripts with low ribosome occupancy, the ER-anchored PARN modulated DNA damage response and thereby cell viability. These findings revealed that a highly regulated communication between mRNA degradation rate and translation efficiency is present on the ER surface in situ and that PARN may contribute to this communication by modulating the dynamic ribosome reallocation between transcripts with low and high ribosome occupancies.

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

2021 ◽  
Vol 118 (48) ◽  
pp. e2106477118
Author(s):  
Shyam Twayana ◽  
Albino Bacolla ◽  
Angelica Barreto-Galvez ◽  
Ruth B. De-Paula ◽  
William C. Drosopoulos ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Single Molecule ◽  
Human Population ◽  
Repetitive Sequences ◽  
Fragile Sites ◽  
Metaphase Chromosomes ◽  
Healthy Human ◽  
Common Fragile Sites ◽  
Dna Structures

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

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