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PubMed2004

UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress.

Ataxia-telangiectasia-mutated and Rad3-related (ATR) plays an essential role in the maintenance of genome integrity and cell viability. The kinase is activated in response to DNA damage and initiates a checkpoint signaling cascade by phosphorylating a number of downstream substrates including Chk1. Unlike ataxia-telangiectasia-mutated (ATM), which appears to be mainly activated by DNA double-strand breaks, ATR can be activated by a variety of DNA damaging agents. However, it is still unclear what triggers ATR activation in response to such diverse DNA lesions. One model proposes that ATR can directly recognize DNA lesions, while other recent data suggest that ATR is activated by a common single-stranded DNA (ssDNA) intermediate generated during DNA repair. In this study, we show that UV lesions do not directly activate ATR in vivo. In addition, ssDNA lesions created during the repair of UV damage are also not sufficient to activate the ATR-dependent pathway. ATR activation is only observed in replicating cells indicating that replication stress is required to trigger the ATR-mediated checkpoint cascade in response to UV irradiation. Interestingly, H2AX appears to be required for the accumulation of ATR at stalled replication forks. Together our data suggest that ssDNA at arrested replication forks recruits ATR and initiates ATR-mediated phosphorylation of H2AX and Chk1. Phosphorylated H2AX might further facilitate ATR activation by stabilizing ATR at the sites of arrested replication forks.

Cell Cycle Proteins↗
PubMed2004

Loss of rereplication control in Saccharomyces cerevisiae results in extensive DNA damage.

To maintain genome stability, the entire genome of a eukaryotic cell must be replicated once and only once per cell cycle. In many organisms, multiple overlapping mechanisms block rereplication, but the consequences of deregulating these mechanisms are poorly understood. Here, we show that disrupting these controls in the budding yeast Saccharomyces cerevisiae rapidly blocks cell proliferation. Rereplicating cells activate the classical DNA damage-induced checkpoint response, which depends on the BRCA1 C-terminus checkpoint protein Rad9. In contrast, Mrc1, a checkpoint protein required for recognition of replication stress, does not play a role in the response to rereplication. Strikingly, rereplicating cells accumulate subchromosomal DNA breakage products. These rapid and severe consequences suggest that even limited and sporadic rereplication could threaten the genome with significant damage. Hence, even subtle disruptions in the cell cycle regulation of DNA replication may predispose cells to the genomic instability associated with tumorigenesis.

Cell Cycle Proteins↗
PubMed2000

Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS untargeted mutagenesis.

When challenged by DNA-damaging agents, Escherichia coli cells respond by inducing the SOS stress response, which leads to an increase in mutation frequency by two mechanisms: translesion replication, a process that causes mutations because of misinsertion opposite the lesions, and an inducible mutator activity, which acts at undamaged sites. Here we report that DNA polymerase V (pol V; UmuC), which previously has been shown to be a lesion-bypass DNA polymerase, was highly mutagenic during in vitro gap-filling replication of a gapped plasmid carrying the cro reporter gene. This reaction required, in addition to pol V, UmuD', RecA, and single-stranded DNA (ssDNA)-binding protein. pol V produced point mutations at a frequency of 2.1 x 10(-4) per nucleotide (2.1% per cro gene), 41-fold higher than DNA polymerase III holoenzyme. The mutational spectrum of pol V was dominated by transversions (53%), which were formed at a frequency of 1.3 x 10(-4) per nucleotide (1. 1% per cro gene), 74-fold higher than with pol III holoenzyme. The prevalence of transversions and the protein requirements of this system are similar to those of in vivo untargeted mutagenesis (SOS mutator activity). This finding suggests that replication by pol V, in the presence of UmuD', RecA, and ssDNA-binding protein, is the basis of chromosomal SOS untargeted mutagenesis.

ATP-Binding Cassette Transporters↗
PubMed2001

Human bone marrow transplant rejection is associated with telomere cleavage.

Telomeres that guard chromosomes are shortened with each cell division because of replication-dependent sequence loss at both termini. The gradual erosion of telomeric length has been directly related to the process of aging in vivo. Recently we have reported, in murine and human cancer cells treated with different apoptogens, cleavage and extrusion of telomeric DNA prior to cell death on one hand and an amplification of telomeric DNA in metastatic epithelial malignancies of different histopathologic origin on the other. This study tested our hypothesis that telomere cleavage is linked to transplant rejection in cancer patients receiving stem cells either from bone marrow (BM) or umbilical cord blood transfusion. Telomere integrity and mitotic catastrophe were studied by cytogenetic and molecular fluorescence in situ hybridization (FISH) techniques in two BM samples taken from a male stem cell transplant recipient diagnosed with aplastic anemia. The first BM sample, which was aspirated 27 days after transplant, was mitotically active. Only one of 50 metaphases showed a chromatid break. Every cell karyotyped was of male origin with 46, XY chromosome constitution. The second BM sample aspirated 52 days after transplant gave no metaphases and most interphase cells appeared dead. FISH preparations of the second BM sample showed cleavage and drastic reduction of telomeric DNA at the time the patient was rejecting the transplant. In contrast, the first BM sample had shown no indication of cleavage of the telomeric DNA, although the percentage of telomeric area was smaller than in the control. The replicative stress imposed on the stem cells engrafted may result in an accelerated aging effect, possibly due to the erosion of telomeric DNA. We, therefore, conclude that BM rejection could be directly associated with the cleavage, clustering, and extrusion of telomeric DNA in the donor cells.

Adolescent↗
PubMed1998

Differential expression of mitochondrial DNA replication factors in mammalian tissues.

Mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication are regulated during development and in response to physiological stresses, but the regulatory events that control the abundance of mtDNA in cells of higher eukaryotes have not been defined at a molecular level. In this study, we observed that expression of the catalytic subunit of DNA polymerase gamma (POLgammaCAT) mRNA varies little among different tissues and is not increased by continuous neural activation of skeletal muscle, a potent stimulus to mitochondrial biogenesis. Increased copy number for the POLgamma locus in a human cell line bearing a partial duplication of chromosome 15 increased the abundance of POLgammaCAT mRNA without up-regulation of mtDNA. In contrast, expression of mitochondrial single-stranded DNA-binding (mtSSB) mRNA is regulated coordinately with variations in the abundance of mtDNA among tissues of mammalian organisms and is up-regulated in association with the enhanced mitochondrial biogenesis that characterizes early postnatal development of the heart and the adaptive response of skeletal myofibers to motor nerve stimulation. In addition, we noted that expression of mtSSB is concentrated within perinuclear mitochondria that constitute active sites of mtDNA replication. We conclude that constitutive expression of the gene encoding the catalytic subunit of mitochondrial DNA polymerase is sufficient to support physiological variations in mtDNA replication among specialized cell types, whereas expression of the mtSSB gene is controlled by molecular mechanisms acting to regulate mtDNA replication or stability in mammalian cells.

Animals↗
PubMed1993

Replication of damaged DNA and the molecular mechanism of ultraviolet light mutagenesis.

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.

DNA Damage↗
PubMed2026

The emerging landscape of polymerase θ in tumor pathogenesis and precision treatment.

DNA polymerase θ (Polθ) has emerged as a central yet paradoxical regulator of genome stability and tumor progression. Unlike conventional DNA repair factors, Polθ governs an error-prone double-strand break repair pathway-termed theta-mediated end joining (TMEJ)-which becomes essential for survival in homologous recombination (HR)-deficient cancers while simultaneously fueling genomic instability. This review presents a comprehensive and updated synthesis of Polθ's structural architecture, regulatory networks, and context-dependent functions across major malignancies, including ovarian, breast, lung, and colorectal cancers. We highlight recently uncovered mechanisms, such as the iron-FTH1/FTL-POLQ-RAD51 axis in platinum-resistant ovarian cancer, the differential POLQ regulation under methionine restriction in BRCA1-mutant breast cancer, and the identification of germline POLQ mutations defining a novel hereditary colorectal cancer subtype. Beyond DNA repair, we integrate emerging evidence linking Polθ to replication stress tolerance, base excision repair, and immune modulation via type I interferon activation and macrophage polarization. The review also provides a critical evaluation of current Polθ inhibitors-including ART558, RP-6685, novobiocin, and AB25583-focusing on their mechanisms, synthetic lethality profiles, and ability to overcome PARP inhibitor resistance. By bridging molecular mechanisms with tumor biology and therapeutic translation, this review offers a unique framework for understanding Polθ as both a prognostic biomarker and a precision oncology target, with implications for combination therapies involving radiotherapy, immunotherapy, and chemotherapy.

Cancer therapy↗
PubMed2003

Epstein-Barr virus can inhibit genotoxin-induced G1 arrest downstream of p53 by preventing the inactivation of CDK2.

Epstein-Barr virus (EBV) is involved in the pathogenesis of several B cell lymphoproliferations, but the precise contribution it makes to the aetiology of each remains unclear. In vitro, the virus has potent growth transforming activity and efficiently induces the continuous proliferation of normal human B cells. A comparison of EBV-infected primary B cells with an isogenic population induced to proliferate by CD40-ligand (CD40L) and IL4 has revealed that EBV can override - by a novel mechanism - the p53/pRb-mediated G1 checkpoint activated in normal B cells by a genotoxic stress. In cells responding to cisplatin, although p53 is stabilized and activated, EBV latent gene expression appears to inhibit the accumulation of newly synthesized p21(WAF1/CIP1) and the downregulation of cyclin D2 that occur in the normal cells. Consequently, in the EBV-infected cells, CDK2 remains active, hyperphosphorylation of pRb is maintained and the replication of damaged DNA can occur. Under conditions of severe genomic stress, this absence of p21(WAF1/CIP1) function can result in apoptosis; however, when damage is less sustained, genomic instability may arise and this in turn could contribute to the development of a variety of EBV-associated B cell malignancies.

B-Lymphocytes↗
PubMed2025

The CGG triplet repeat binding protein 1 counteracts R-loop induced transcription-replication stress.

The CGG triplet repeat binding protein 1 (CGGBP1) binds to CGG repeats and has several important cellular functions, but how this DNA sequence-specific binding factor affects transcription and replication processes is an open question. Here, we show that CGGBP1 binds human gene promoters containing short (<&#x2009;5) CGG-repeat tracts prone to R-loop formation. Loss of CGGBP1 leads to deregulated transcription, transcription-replication-conflicts (TRCs) and accumulation of Serine-5 phosphorylated RNA polymerase II (RNAPII), indicative of promoter-proximal stalling and a defect in transcription elongation. Consistently, an episomal CGG-repeat-containing model locus as well as endogenous genes show deregulated transcription, R-loop accumulation and increased RNAPII chromatin occupancy in CGGBP1-depleted cells. We identify the DEAD-box RNA:DNA helicases DDX41 and DHX15 as interaction partners specifically recruited by CGGBP1. Co-depletion experiments show that DDX41 and CGGBP1 work in the same pathway to unwind R-loops and avoid TRCs. Together, our work shows that short trinucleotide repeats are a source of genome-destabilizing secondary structures, and cells rely on specific DNA-binding factors to maintain proper transcription and replication coordination at short CGG repeats.

Humans↗
PubMed2006

Human topoisomerase I forms double cleavage complexes on natural DNA.

DNA topoisomerase I releases torsional stress generated in chromatin during transcription and replication. Usually topoisomerase I is recognized to work as a monomer, but previously we have shown that two molecules can form a dimer-like protein-protein complex on a 'suicide' DNA substrate resulting in a topoisomerase I double cleavage complex. Here we show that during the normal relaxation reaction a considerable fraction of human topoisomerase I formed transient dimers on plasmid DNA too. Recombinant as well as topoisomerase I purified from human cells formed double cleavage complexes within a distance of 12 or 14 nucleotides. When topoisomerase I was isolated from camptothecin-treated HeLa cells, a considerable fraction migrated to the same position as topoisomerase I bearing a covalently bound 12-to-14-mer oligonucleotide. Taken together our data suggest that human topoisomerase I double cleavage complexes are part of the normal catalytic cycle of this enzyme that occur in vitro and possibly also in vivo.

Camptothecin↗
PubMed2006

MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens.

MSH2 is a central component of the mismatch repair pathway that targets mismatches arising during DNA replication, homologous recombination (HR) and in response to genotoxic stresses. Here, we describe the function of MSH2 in the moss Physcomitrella patens, as deciphered by the analysis of loss of function mutants. Ppmsh2 mutants display pleiotropic growth and developmental defects, which reflect genomic instability. Based on loss of function of the APT gene, we estimated this mutator phenotype to be at least 130 times higher in the mutants than in wild type. We also found that MSH2 is involved in some but not all the moss responses to genotoxic stresses we tested. Indeed, the Ppmsh2 mutants were more tolerant to cisplatin and show higher sensitivity to UV-B radiations. PpMSH2 gene involvement in HR was studied by assessing gene targeting (GT) efficiency with homologous and homeologous sequences. GT efficiency with homologous sequences was slightly decreased in the Ppmsh2 mutant compared with wild type. Strikingly GT efficiency with homeologous sequences decreased proportionally to sequence divergence in the wild type whereas it remained unaffected in the mutants. Those results demonstrate the role of PpMSH2 in the maintenance of genome integrity and in homologous and homeologous recombination.

Bryopsida↗
PubMed2006

Environmental stress and lesion-bypass DNA polymerases.

In nature, microbes live under a variety of harsh conditions, such as excess DNA damage, starvation, pH shift, or high temperatures. Microbial cells respond to such stressful conditions mostly by switching global patterns of gene expression to relieve the environmental stress. The SOS response, which is induced by DNA damage, is one such global network of gene expression that plays a crucial role in balancing the genomic stability and flexibility that are necessary to adapt to harsh environments. Here, I review the roles of SOS-inducible and noninducible lesion-bypass DNA polymerases in mutagenesis induced by environmental stress, and discuss how these polymerases are coordinated for the replication of damaged chromosomes. Possible contributions of lesion-bypass DNA polymerase in hyperthermophilic archaea, e.g., Sulfolobus solfataricus, to genome maintenance are also discussed.

Archaea↗
PubMed1998

Effect of water stress on cell division and cell-division-cycle 2-like cell-cycle kinase activity in wheat leaves

In wheat (Triticum aestivum) seedlings subjected to a mild water stress (water potential of -0.3 MPa), the leaf-elongation rate was reduced by one-half and the mitotic activity of mesophyll cells was reduced to 42% of well-watered controls within 1 d. There was also a reduction in the length of the zone of mesophyll cell division to within 4 mm from the base compared with 8 mm in control leaves. However, the period of division continued longer in the stressed than in the control leaves, and the final cell number in the stressed leaves reached 85% of controls. Cyclin-dependent protein kinase enzymes that are required in vivo for DNA replication and mitosis were recovered from the meristematic zone of leaves by affinity for p13(suc1). Water stress caused a reduction in H1 histone kinase activity to one-half of the control level, although amounts of the enzyme were unaffected. Reduced activity was correlated with an increased proportion of the 34-kD Cdc2-like kinase (an enzyme sharing with the Cdc2 protein of other eukaryotes the same size, antigenic sites, affinity for p13(suc1), and H1 histone kinase catalytic activity) deactivated by tyrosine phosphorylation. Deactivation to 50% occurred within 3 h of stress imposition in cells at the base of the meristematic zone and was therefore too fast to be explained by a reduction in the rate at which cells reached mitosis because of slowing of growth; rather, stress must have acted more immediately on the enzyme. The operation of controls slowing the exit from the G1 and G2 phases is discussed. We suggest that a water-stress signal acts on Cdc2 kinase by increasing phosphorylation of tyrosine, causing a shift to the inhibited form and slowing cell production.

Journal Article↗
PubMed2003

MutY is down-regulated by oxidative stress in E. coli.

In Escherichia coli, MutM (8-oxoG DNA glycosylase/lyase or Fpg protein), MutY (adenine DNA glycosylase) and MutT (8-oxodGTPase) function cooperatively to prevent mutation due to 7, 8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic oxidative DNA adduct. MutM activity has been demonstrated to be induced by oxidative stress. Its regulation is under the negative control of the global regulatory genes, fur, fnr and arcA. However, interestingly the presence of MutY increases the mutation frequency in mutT- background because of MutY removes adenine (A) from 8-oxoG:A which arises from the misincorporation of 8-oxoG against A during DNA replication. Accordingly we hypothesized that the response of MutY to oxidative stress is opposite to that of MutM and compared the regulation of MutY activity with MutM under various oxidative stimuli. Unlike MutM, MutY activity was reduced by oxidative stress. Its activity was reduced to 30% of that of the control when E. coli was treated with paraquat (0.5mM) or H2O2 (0.1 mM) and induced under anaerobic conditions to more than twice that observed under aerobic conditions. The reduced mRNA level of MutY coincided with its reduced activity by paraquat treatment. Also, the increased activity of MutY in anaerobic conditions was reduced further in E. coli strains with mutations in fur, fnr and arcA and the maximum reduction in activity was when all mutations were present in combination, indicating that MutY is under the positive control of these regulatory genes. Therefore, the down-regulation of MutY suggests that there has been complementary mechanism for its mutagenic activity under special conditions. Moreover, the efficacy of anti-mutagenic action should be enhanced by the reciprocal co-regulation of MutM.

DNA↗
PubMed2006

[Genetic determinants of childhood leukemia].

Pediatric cancers affect approximately 1 in every 500 children before the age of 15. Little is known about the etiology of this heterogeneous group of diseases despite the fact they constitute the major cause of death by disease among this population. Because of its relatively high prevalence, most of the work done in pediatric oncogenetics has been focused on leukemias, particularly acute lymphoblastic leukemia (ALL). Although it is now well accepted that genetic variations play a significant role in determining individual's cancer susceptibility, few studies have explored genetic susceptibility to childhood leukemia with respect to common polymorphisms. The biochemical and genetic mechanisms contributing to cancer susceptibility are numerous and can be grouped into broad categories: 1) cellular growth and differentiation, 2) DNA replication and repair, 3) metabolism of carcinogens, 4) apoptosis, 5) oxidative stress response and 6) cell cycle. To evaluate whether candidate genes in these pathways are involved in childhood leukemogenesis, we conducted association studies. We showed that leukemogenesis in children may be associated with DNA variants in some of these genes and that the combination of genotypes seems to be more predictive of risk then either of them independently. We also observed that, at least at some loci, the parental genetics might be important in predicting the risk of cancer in this pediatric model of a complex disease. Taken together, these results indicate that the investigation of a single enzyme and/or a single genotype might not be sufficient to explain the etiology of childhood leukemia because of the complexity of the environment and that of the inter-individual variability in cancer susceptibility.

Adolescent↗
PubMed2005

Telomeres as biomarkers for ageing and age-related diseases.

Telomeres in telomerase-negative cells shorten during DNA replication in vitro due to numerous causes including the inability of DNA polymerases to fully copy the lagging strand, DNA end processing and random damage, often caused by oxidative stress. Short telomeres activate replicative senescence, an irreversible cell cycle arrest. Thus, telomere length is an indicator of replicative history, of the probability of cell senescence, and of the cumulative history of oxidative stress. Telomeres in most human cells shorten during ageing in vivo as well, suggesting that telomere length could be a biomarker of ageing and age-related morbidity. There are two distinct possibilities: First, in a tissue-specific fashion, short telomeres might indicate senescence of (stem) cells, and this might contribute to age-related functional attenuation in this tissue. Second, short telomeres in one tissue might cause systemic effects or might simply indicate a history of high stress and damage in the individual and could thus act as risk markers for age-related disease residing in a completely different tissue. In recent years, data have been published to support both approaches, and we will review these. While they together paint a fairly promising picture, it needs to be pointed out that until now most of the evidence is correlative, that much of it comes from underpowered studies, and that causal evidence for essential pathways, for instance for the impact of cell senescence on tissue ageing in vivo, is still very weak.

Aging↗
PubMed2006

Challenges identifying genetic determinants of pediatric cancers--the childhood leukemia experience.

Pediatric cancers affect approximately 1 in every 500 children before the age of 15. Little is known about the etiology of this heterogeneous group of diseases despite the fact they constitute the major cause of death by disease among this population. Because of its relatively high prevalence, most of the work done in pediatric oncogenetics has been focused on leukemias, particularly acute lymphoblastic leukemia (ALL). Although it is now well accepted that genetic variation plays a significant role in determining individual's cancer susceptibility, few studies have explored genetic susceptibility to childhood leukemia with respect to common polymorphisms. The biochemical and genetic mechanisms contributing to cancer susceptibility are numerous and can be grouped into broad categories: (1) cellular growth and differentiation, (2) DNA replication and repair, (3) metabolism of carcinogens (4) apoptosis, (5) oxidative stress response and (6) cell cycle. To evaluate whether candidate genes in these pathways are involved in childhood leukemogenesis, we conducted case-control studies. We showed that leukemogenesis in children may be associated with DNA variants in some of these genes and that the combination of genotypes seems to be more predictive of risk than either of them independently. We also observed that, at least at some loci, the parental genetics might be important in predicting the risk of cancer in this pediatric model of a complex disease. Taken together, these results indicate that the investigation of a single enzyme and/or a single genotype might not be sufficient to explain the etiology of childhood leukemia because of the complexity of the environment and that of the inter-individual variability in cancer susceptibility.

Child↗