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PubMed2003

Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes.

The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase 1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2'-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novo synthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such as NF-kappaB, JunB, ATF-3, and GADD45beta (growth arrest DNA damage 45beta gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.

Cell Division↗
PubMed2006

Depletion of CHK1, but not CHK2, induces chromosomal instability and breaks at common fragile sites.

Common fragile sites are specific regions of the genome that form gaps and breaks on metaphase chromosomes when DNA synthesis is partially inhibited. Fragile sites and their associated genes show frequent deletions and other rearrangements in cancer cells, and may be indicators of DNA replication stress early in tumorigenesis. We have previously shown that the DNA damage response proteins ATR, BRCA1 and FANCD2 play critical roles in maintaining the stability of fragile site regions. To further elucidate the pathways regulating fragile site stability, we have investigated the effects of depletion of the cell cycle checkpoint kinases, CHK1 and CHK2 on common fragile site stability in human cells. We demonstrate that both CHK1 and CHK2 are activated following treatment of cells with low doses of aphidicolin that induce fragile site breakage. Furthermore, we show that depletion of CHK1, but not CHK2, using short-interfering RNA (siRNA) leads to highly destabilized chromosomes and specific common fragile site breakage. In many cells, CHK1 depletion resulted in extensive chromosome fragmentation, which was distinct from endonucleolytic cleavage commonly associated with apoptosis. These findings demonstrate a critical role for the CHK1 kinase in regulating chromosome stability, and in particular, common fragile site stability.

Cells, Cultured↗
PubMed2025

Lamin A/C loss promotes R-loop-mediated genomic instability and poor survival in small-cell lung cancer.

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [W. Xie et al., Curr. Biol. 26, 2651-2658 (2016)]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [S. Graziano et al., Nucleus 9, 258-275 (2018)]. Here, we define a mechanistic role for LMNA in preserving genome stability in small-cell lung cancer (SCLC), a malignancy marked by extreme genomic instability [N. Takahashi et al., Cancer Res. Commun. 2, 503-517 (2022)]. LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA deficiency disrupts nuclear pore complex organization, specifically reducing phenylalanine-glycine (FG)-nucleoporin incorporation, resulting in impaired RNA export and nuclear retention of RNA. LMNA expression is repressed by EZH2 and reexpressed during SCLC differentiation from neuroendocrine (NE) to non-NE states, and low LMNA levels correlate with poor clinical outcomes. These findings establish LMNA as a key regulator of nuclear transport and genome integrity, linking nuclear architecture to SCLC progression and therapeutic vulnerability.

Lamin Type A↗
PubMed2004

DNA replication defects, spontaneous DNA damage, and ATM-dependent checkpoint activation in replication protein A-deficient cells.

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2023

DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation.

The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.

DNA Replication↗
PubMed2026

G-quadruplex upstream of PAX9 TSS acts as a 3D-genome scaffold to remotely silence X-linked genes and modulate cell-cycle progression.

G-quadruplexes (G4s) are non-canonical DNA secondary structures that act as local replication barriers and transcriptional regulators. Whether G4 can simultaneously influence splicing, DNA replication, and long-range, trans-chromosomal gene regulation remains untested. Here we combined in vitro biophysics, CRISPR mutagenesis and multi-omics to dissect a conserved G4 motif (QS1) located ~173 bp upstream of the PAX9 transcription start site. CD spectroscopy confirmed that the wild-type, but not the G-to-T mutant sequence, folds into a stable parallel G4 under physiological K+. In human cells, disruption of the QS1 G4 changed chromatin accessibility, remotely down-regulated a cohort of X-linked genes, accelerated migration and delayed G1/S progression. Integrative analysis of ATAC-seq, RNA-seq profiling reveals that the QS1 G4 acts as a three-dimensional genome scaffold linking PAX9 to cell-cycle and metabolic networks. Our findings establish a pleiotropic role for a single promoter G4 in coordinating DNA replication stress, chromatin architecture and trans-chromosomal transcriptional control.

G-Quadruplexes↗
PubMed1998

Linkage of ATM to cell cycle regulation by the Chk2 protein kinase.

In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators. We identified Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases required for the DNA damage and replication checkpoints. Chk2 was rapidly phosphorylated and activated in response to replication blocks and DNA damage; the response to DNA damage occurred in an ataxia telangiectasia mutated (ATM)-dependent manner. In vitro, Chk2 phosphorylated Cdc25C on serine-216, a site known to be involved in negative regulation of Cdc25C. This is the same site phosphorylated by the protein kinase Chk1, which suggests that, in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis.

Amino Acid Sequence↗
PubMed2006

ATRIP associates with replication protein A-coated ssDNA through multiple interactions.

The ATR (ATM- and rad3-related)-mediated checkpoint pathway has a crucial role in regulating the cellular responses to DNA damage and DNA-replication stress. ATRIP (ATR-interacting protein), the regulatory partner of ATR, binds directly to replication protein A (RPA)-coated ssDNA and enables the ATR-ATRIP complex to recognize this DNA damage-induced structure. Here, we show that ATRIP associates with RPA-ssDNA through multiple interactions. Two major RPA-ssDNA-interacting domains of ATRIP were mapped to the regions flanking the conserved coiled-coil domain. In contrast to a recent article, we found that ATRIP mutants lacking the N terminus retained the ability to bind to RPA-ssDNA, suggesting that the multiple interactions between ATRIP and RPA-ssDNA may function redundantly in the recruitment of ATR-ATRIP. Unexpectedly, one internal region of ATRIP exhibited affinity to ssDNA, suggesting that ATRIP may interact with ssDNA in the ATRIP-RPA-ssDNA complex. Also, the N terminus of ATRIP associated with RPA-ssDNA in two distinct ways, indicating a dynamic and regulated association between ATRIP and RPA-ssDNA.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Formation of a complex between nucleolin and replication protein A after cell stress prevents initiation of DNA replication.

We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin-hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin-hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.

Cell Compartmentation↗
PubMed2025

Lamin A/C Deficiency Drives Genomic Instability and Poor Survival in Small-Cell Lung Cancer through Increased R-loop Accumulation.

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [1]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [2]. Here, we investigate LMNA's role in small-cell lung cancer (SCLC), a highly aggressive malignancy characterized by extreme genomic instability [3, 4]. We demonstrate that LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA loss disrupts nuclear pore complex distribution, reducing phenylalanine-glycine (FG)-nucleoporin incorporation and impairing RNA export efficiency. Furthermore, we show that LMNA expression is epigenetically repressed by EZH2 during SCLC differentiation from neuroendocrine (NE) to non-NE states. Clinically, low LMNA levels correlate with significantly worse survival in SCLC patients. These findings uncover a novel role for LMNA in safeguarding genome integrity and shaping tumor heterogeneity, with broad implications for cancer and aging.

Biological Sciences↗
PubMed1990

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress.

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.

Animals↗
PubMed2026

Plasticity of hepatic metabolism in Arctic char (Salvelinus alpinus) in response to cyclic hypoxia.

The emergence of cyclic hypoxia puts aquatic organisms' homeostasis under significant strain. Energetic metabolism as well as protein synthesis and folding are particularly altered during hypoxia, while reoxygenation imposes an oxidative challenge. Currently, little is known about how hypoxia-sensitive organisms respond to large oxygen fluctuations. Our previous work on Arctic char revealed that this salmonid, despite being strongly affected by acute hypoxia and reoxygenation (H/R), can successfully reestablish homeostasis, notably through adjustments to hepatic mitochondrial metabolism. However, the mechanisms underlying this acclimation remain poorly understood. We hypothesized that Arctic char remodel their hepatic proteome to optimize energy metabolism, reorganize oxygen-demanding pathways, and maintain cellular homeostasis during repeated H/R cycles. By exposing Arctic char to two or fifteen days of diel cyclic hypoxia, we confirmed this species' limited capacity to respond to acute H/R. Nevertheless, after fifteen cycles, fish adjusted their energetic metabolism through coordinated regulation of carbohydrate and lipid pathways and upregulation of amino acid metabolism. Mitochondrial metabolism was strongly reorganized, particularly at the ubiquinone-Complex III interaction level, alongside adjustments in proline utilization and protein processing. Moreover, protein processing and folding pathways were stimulated in both mitochondria and the endoplasmic reticulum. However, chronic cyclic hypoxia may still promote non-mitochondrial ROS production, DNA replication stress, and impaired immune function. This study highlights how a hypoxia-sensitive fish progressively reorganizes its metabolism and oxygen-demanding pathways to establish a phenotype adapted to chronic cyclic hypoxia, while also revealing the physiological costs associated with this acclimation.

Animals↗
PubMed2025

Replication stress increases de novo CNVs across the malaria parasite genome.

Changes in the copy number of large genomic regions, termed copy number variations (CNVs), contribute to important phenotypes. CNVs are readily identified using conventional approaches when present in a large fraction of the cell population. However, CNVs in only a few genomes are often overlooked but important; if beneficial, a de novo CNV that arises in a single genome can expand during selection to create a population of cells with novel characteristics. While single cell methods for studying de novo CNVs are increasing, we continue to lack information about CNV dynamics in rapidly evolving microbial populations. Here, we investigated de novo CNVs in the genome of the Plasmodium parasite that causes human malaria. The highly AT-rich P. falciparum genome readily accumulates CNVs that facilitate rapid adaptation. We employed low-input genomics and specialized computational tools to evaluate the impact of sub-lethal stress on the de novo CNV rate. We observed a significant increase in genome-wide de novo CNVs following treatment with an antimalarial compound that inhibits replication. De novo CNVs encompassed genes from various cellular pathways participating in human infection. This snapshot of CNV dynamics emphasizes the connection between replication stress, DNA repair, and CNV generation in this important microbial pathogen.

Journal Article↗
PubMed2005

ATR activation necessary but not sufficient for p53 induction and apoptosis in hydroxyurea-hypersensitive myeloid leukemia cells.

Hydroxyurea (HU) is a competitive inhibitor of ribonucleotide reductase that is used for the treatment of myeloproliferative disorders. HU inhibits DNA replication and induces apoptosis in a cell type-dependent manner, yet the relevant pathways that mediate apoptosis in response to this agent are not well characterized. In this study, we employed the human myeloid leukemia 1 (ML-1) cell line as a model to investigate the mechanisms of HU-induced apoptosis. Exposure of ML-1 cells to HU caused rapid cell death that was accompanied by hallmark features of apoptosis, including membrane blebbing, phosphatidylserine translocation, and caspase activation. HU-induced apoptosis required new protein synthesis, was induced by HU exposures as short as 15 min, and correlated with the accumulation of p53 and induction of the p53 target gene PUMA. p53 induction in ML-1 cells was ATR dependent and downregulation of p53 through RNAi delayed HU-induced apoptosis. HU did not induce p53 or induce apoptosis in Molt-3 leukemia cells, even though exposure to HU induced a comparable level of DNA damage and robustly activated the ATR pathway. The microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event contributes to p53 induction and apoptosis in this cell line. Our findings outline an HU-induced cell death pathway and suggest that activation of ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.

Apoptosis↗
PubMed1998

p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases.

p21waf1 has been shown to mediate the p53-dependent growth arrest induced by DNA-damaging agents. Several functions have been ascribed to p21waf1 that could be involved in this growth arrest. For one, p21waf1 is an efficient inhibitor of cyclin-dependent kinases (CDKs). Also, p21waf1 can interact with proliferating cell nuclear antigen (PCNA), and as such inhibit in vitro DNA-replication. Finally, p21waf1 has been reported to inhibit stress-activated protein kinases (SAPKs). In order to study these multiple functions of p21waf1 we have established U2OS-derived cell lines, in which the expression of p21waf1 can be regulated by the concentration of tetracycline in the culture medium. We observed a virtually complete, but reversible inhibition of cell growth upon induction of p21waf1-expression. Both [3H]thymidine-incorporation and CDK2-activity were strongly inhibited by p21waf1. Upon induction of p21waf1 cells accumulated with a 2N or 4N DNA content suggesting events in G1 and G2 can be inhibited by p21waf1. Indeed, kinase activity associated with cyclin B was reduced dramatically upon induction of p21waf1, although cyclin B continues to be expressed. In contrast, p21waf1 does not seem to inhibit the function of PCNA in ongoing DNA replication, since cells expressing high levels of p21waf1 apparently progressed normally through S-phase. Also, the activity of SAPKs was not substantially affected by the high levels of p21waf1. We conclude that, at least in these U2OS-derived cells, p21waf1 functions as an inhibitor of CDK-activity in G1 and G2, but not as an inhibitor of PCNA or SAPKs.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Pleiotropic resistance to DNA-interactive drugs is associated with increased expression of genes involved in DNA replication, repair, and stress response.

A combination of four genetic suppressor elements (GSEs), two of which are derived from putative transcriptional regulators, was previously found to increase resistance to drugs inhibiting DNA replication in HT1080 fibrosarcoma cells. In the present study, two GSE-transduced cell lines, isolated with and without cytotoxic selection, were found to be resistant to a diverse group of DNA-interactive agents, including aphidicolin, hydroxyurea, cytarabine, etoposide, doxorubicin, and mafosfamide. Changes in gene expression associated with GSE-induced drug resistance were analyzed by cDNA array hybridization and reverse transcription-PCR. Twenty genes were found to be up-regulated in both of the resistant cell lines. These include genes involved in DNA replication and repair (e.g., PCNA, XRCC1, B-MYB, and GADD45), transcriptional regulators associated with stress response, and cell cycle checkpoint control (e.g., YB-1, DBPA, and ATF4), and genes for signal transduction proteins (e.g., protein tyrosine phosphatase 1B and regulatory subunits alpha and beta of cAMP-dependent protein kinase). The observed changes in gene expression may play a role in pleiotropic resistance to different classes of DNA-targeting drugs.

Antineoplastic Agents↗
PubMedDate not supplied

[Suppression of replication and activation of DNA reparative synthesis in stress and prevention of these phenomena by preliminary adaptation].

Effects of preliminary adaptation to short-term stress or to regular hypoxia on disturbances of DNA biosynthesis were studied in liver and heart tissues under conditions of emotional-painful stress (EPS). EPS was found to induced activation of DNA reparative synthesis in heart and liver tissues and affected dissimilarly DNA replication in these tissues: activation of the reaction in heart and suppression in liver tissue. Adaptation to regular hypoxia limited distinctly the burst of DNA reparative synthesis in cells of both these tissues, reduced activation of the DNA replicative synthesis in heart and prevented the stress induced depression of DNA replication in hepatic cell nuclei and mitochondria. Mechanisms of the hepatoprotective effect of adaptation to hypoxia is discussed.

Adaptation, Physiological↗
PubMed2005

DNA damage and replication stress induced transcription of RNR genes is dependent on the Ccr4-Not complex.

Genetic experiments have indicated a role for the Ccr4-Not complex in the response to hydroxyurea (HU) induced replication stress and ionizing radiation in yeast. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4 and degradation of its inhibitor, Sml1p. The Ccr4-Not complex has originally been described as a negative regulator of RNA polymerase II (pol II) transcription, but it has also been implicated in mRNA turnover and protein ubiquitination. We investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU induced Sml1p degradation. We show, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or methyl-methane sulfonate (MMS) treatment. Chromatin immunoprecipitation (ChIP) experiments show that TBP, pol II and Set1p recruitment to the activated RNR3 locus is defective in cells lacking NOT4. Moreover, RNR3-promoter activity is not induced by HU in these cells. Our experiments show that induction of RNR gene transcription is defective in ccr4-not mutant strains, providing an explanation for their sensitivity to HU.

Base Sequence↗