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At least 127 records · Page 7Linked to original sources
PubMed2024

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication.

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.

Animals↗
PubMed2005

The DNA damage response during DNA replication.

Eukaryotic chromosome replication is mediated by multiple replicons and is coordinated with sister chromatid cohesion, DNA recombination, transcription and cell cycle progression. Replication forks stall or collapse at DNA lesions or problematic genomic regions, and these events have often been associated with recombination and chromosomal rearrangements. Stalled forks generate single-stranded DNA that activates the replication checkpoint, which in turn functions to protect the stability of the fork until the replication can resume. Recombination-mediated and damage-bypass processes are the main mechanisms responsible for replication restart. New findings have helped to unmask the molecular mechanisms that sense replication stress, control the stability of replication forks, and regulate the mechanisms that promote replication restart, thereby giving us a better understanding of how genome integrity is preserved during replication.

DNA Damage↗
PubMed2005

Genotoxic stress targets human Chk1 for degradation by the ubiquitin-proteasome pathway.

The Chk1 kinase is a major effector of S phase checkpoint signaling during the cellular response to genotoxic stress. Here, we report that replicative stress induces the polyubiquitination and degradation of Chk1 in human cells. This response is triggered by phosphorylation of Chk1 at Ser-345, a known target site for the upstream activating kinase ATR. The ubiquitination of Chk1 is mediated by E3 ligase complexes containing Cul1 or Cul4A. Treatment of cells with the anticancer agent camptothecin (CPT) triggers Chk1 destruction, which blocks recovery from drug-induced S phase arrest and leads to cell death. These findings indicate that ATR-dependent phosphorylation of Chk1 delivers a signal that both activates Chk1 and marks this protein for proteolytic degradation. Proteolysis of activated Chk1 may promote checkpoint termination under normal conditions, and may play an important role in the cytotoxic effects of CPT and related anticancer drugs.

Bromodeoxyuridine↗
PubMed2006

Fusion tyrosine kinases: a result and cause of genomic instability.

Reciprocal chromosomal translocations may arise as a result of unfaithful repair of spontaneous DNA double-strand breaks, most probably induced by oxidative stress, radiation, genotoxic chemicals and/or replication stress. Genes encoding tyrosine kinases are targeted by these mechanisms resulting in the generation of chimera genes encoding fusion tyrosine kinases (FTKs). FTKs display transforming activity owing to their constitutive kinase activity causing deregulated proliferation, apoptosis, differentiation and adhesion. Moreover, FTKs are able to facilitate DNA repair, prolong activation of G(2)/M and S cell cycle checkpoints, and elevate expression of antiapoptotic protein Bcl-X(L), making malignant cells less responsive to antitumor treatment. FTKs may also stimulate the generation of reactive oxygen species and enhance spontaneous DNA damage in tumor cells. Unfortunately, FTKs compromise the fidelity of DNA repair mechanisms, which contribute to the accumulation of additional genetic abnormalities leading to the resistance to inhibitors such as imatinib mesylate and malignant progression of the disease.

Animals↗
PubMed2026

FANCM is required for the PAX3::FOXO1-driven oncogenic program in rhabdomyosarcoma.

Many cancers are driven by mutationally altered transcription factors (TFs) that rewire cells to an oncogenic state. Cells must activate specific mechanisms to tolerate the burden of oncogenic TF activity. To define such mechanisms, we focused on a canonical oncogenic fusion protein-driven cancer, alveolar rhabdomyosarcoma (ARMS), where the PAX3::FOXO1 fusion protein hyperactivates and mislocalizes PAX3 and FOXO1 TF functions. Employing sequential functional genomic CRISPR-Cas9 screens, we identified FANCM, a DNA translocase in the Fanconi anemia pathway, as a selective dependency in PAX3::FOXO1+ ARMS. FANCM loss reduces fusion protein levels, induces myogenic differentiation, and disrupts the PAX3::FOXO1 transcriptional program, thereby halting oncogenic proliferation. Mechanistically, FANCM depletion exacerbates replication stress (RS) and DNA damage signaling, with chromatin-associated RS enriched at PAX3::FOXO1 target gene loci, resulting in selective downregulation of the oncogenic program. CRISPR exon-tiling screens prioritized FANCM's helicase and DNA-binding domains as essential for this dependency, linking FANCM-mediated replication fork binding to sustained oncogenesis.

ARMS↗
PubMed2000

Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1.

Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.

Amino Acid Sequence↗
PubMed2004

Damage signaling: RecQ sends an SOS to you.

The DNA helicase RecQ is required for proper induction of the SOS response to replication stress in Escherichia coli. Unwinding of stalled replication forks by RecQ family helicases in bacteria, and possibly in eukaryotes, may provide a means of damage signaling and recovering stalled replication forks.

Adenosine Triphosphatases↗
PubMed1998

The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor.

We have identified the yeast CRT1 gene as an effector of the DNA damage and replication checkpoint pathway. CRT1 encodes a DNA-binding protein that recruits the general repressors Ssn6 and Tup1 to the promoters of damage-inducible genes. Derepression of the Crt1 regulon suppresses the lethality of mec1 and rad53 null alleles and is essential for cell viability during replicative stress. In response to DNA damage and replication blocks, Crt1 becomes hyperphosphorylated and no longer binds DNA, resulting in transcriptional induction. CRT1 is autoregulated and is itself induced by DNA damage, indicating the existence of a negative feedback pathway that facilitates return to the repressed state after elimination of damage. The inhibition of an autoregulatory repressor in response to DNA damage is a strategy conserved throughout prokaryotic and eukaryotic evolution.

Animals↗
PubMed2006

The WTM genes in budding yeast amplify expression of the stress-inducible gene RNR3.

Cellular responses to DNA damage and inhibited replication are evolutionarily conserved sets of pathways that are critical to preserving genome stability. To identify new participants in these responses, we undertook a screen for regulators that, when present on a high-copy vector, alter expression of a DNA damage-inducible RNR3-lacZ reporter construct in Saccharomyces cerevisiae. From this screen we isolated a plasmid encoding two closely related paralogs, WTM1 and WTM2, that greatly increases constitutive expression of RNR3-lacZ. Moderate overexpression of both genes together, or high-level expression of WTM2 alone from a constitutive promoter, upregulates RNR3-lacZ in the absence of DNA damage. Overexpressed, tagged Wtm2p is associated with the RNR3 promoter, indicating that this effect is likely direct. Further investigation reveals that Wtm2p and Wtm1p, previously described as regulators of meiotic gene expression and transcriptional silencing, amplify transcriptional induction of RNR3 in response to replication stress and modulate expression of genes encoding other RNR subunits.

Chromatin Immunoprecipitation↗
PubMed2024

Hepatitis B Virus-KMT2B Integration Drives Hepatic Oncogenic Processes in a Human Gene-edited Induced Pluripotent Stem Cells-derived Model.

BACKGROUND & AIMS: Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC; however, its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cell (iPSCs) model. METHODS: Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). RESULTS: The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation (H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. CONCLUSIONS: Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Humans↗
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↗
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↗
PubMed2003

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1.

The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis.

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

Cell Cycle↗
PubMedDate not supplied

FHA domain-mediated DNA checkpoint regulation of Rad53.

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.

Cell Cycle Proteins↗
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↗
PubMed2004

Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling.

Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.

Aphidicolin↗