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Results for “DNA Replication Stress”

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At least 541 records · Page 30Linked to original sources
PubMed2001

Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity.

The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress-inducing agents is the subject of much research. We have investigated the relationship between heat-induced damage to DNA replication machinery and the lethal effects of heat shock, in S-phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S-phase, can rescue S-phase HeLa cells from the lethal effects of heat shock. When S-phase, HeLa cells were held for 5-6 h in 3 microM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co-isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S-phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S-phase correlated with cell killing. Heat-induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat-induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S-phase progression but are repairable if S-phase progression is delayed.

Aphidicolin↗
PubMed1991

Stress relaxation of contracted collagen gels: disruption of actin filament bundles, release of cell surface fibronectin, and down-regulation of DNA and protein synthesis.

Relaxation of stressed collagen gels provides a model system uniquely suited to studying the regulation of cell morphology and biosynthetic function by tissue organization. Stress relaxation results in rapid, synchronous changes in cell morphology without enzymatic or other drug treatments, and makes possible an analysis of the initial cellular events associated with changes in tissue organization. During the first hour after stress relaxation, we observed transient hypercontraction of collagen gels and loss of collagen fibril organization as stress in the system dissipated. Morphological changes in the fibroblasts included retraction of pseudopodia, collapse of cytoplasmic actin filament bundles, and loss of cell surface fibronectin. Accompanying these morphological changes, we observed marked decreases in DNA and protein synthesis, especially of fibronectin and type I procollagens. These results show that changes in tissue organization can exert rapid and profound effects on the morphology and biosynthetic function of cells within the tissue.

Actin Cytoskeleton↗
PubMed2006

Model for forward polymerization and switching transition between polymerase and exonuclease sites by DNA polymerase molecular motors.

Based on the available crystal structure a model is presented for the polymerization activity and switching transition between polymerase and exonuclease sites of a DNA polymerase molecular motor. Using the model, the fast polymerization rate for correctly base-paired DNA and much reduced polymerization rate after an incorporation of a mismatched base can be well explained. The dependences of the polymerization rate and exonuclease rate on mechanical tension acting on the DNA template are studied. The switching rates between the two sites are analyzed. All the results show good quantitative agreement with the available experimental results.

Base Pairing↗
PubMed2026

Distinct functions of mammalian RAD51 paralogs in genome maintenance.

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are evolutionarily conserved essential proteins for cell survival and genome maintenance. RAD51 paralogs were originally identified to play a role in homologous recombination-mediated repair of DNA double-strand breaks (DSBs). However, investigations over the last decade have uncovered new roles of RAD51 paralogs beyond DSB repair in replication stress responses, including replication fork progression, fork stability, and its restart. Recent structural studies have not only uncovered the molecular architecture of previously known RAD51 paralog complexes but also identified novel paralog complex assemblies, providing mechanistic insights into their various genome-maintenance functions. Additionally, a role for RAD51 paralogs in resolving R-loops has been identified, and studies with cancer-associated variants suggest that RAD51 paralogs are potential determinants of cancer susceptibility and therapeutic responses. In the present review, we highlight the recently deciphered structures and novel functions of RAD51 paralog complexes and discuss the clinical and therapeutic implications.

Rad51 Recombinase↗
PubMed2026

When R-Loops Go Awry: Genome Instability and Neurological Diseases.

The basic structure of DNA is a double helix formed by base pairing between complementary strands. However, during transcription, RNA hybridizes with the template DNA, whereas the complementary DNA strand becomes displaced and remains unpaired. This process forms a DNA-RNA hybrid structure known as an R-loop; similar structures can also occur in a non-co-transcriptional manner. In recent years, R-loops have been reported to be involved in various cellular functions. However, when not properly regulated, they can compromise genomic DNA stability. R-loops play roles in gene expression, DNA replication, and transcription termination. Dysregulation of R-loop homeostasis has been implicated in various human diseases, including neurological diseases. In this review, we discuss the physiological and pathological roles of R-loops, their related regulatory mechanisms controlling their formation and resolution, and their association with neurological diseases.

Humans↗
PubMed2006

Cellular senescence in aging primates.

The aging of organisms is characterized by a gradual functional decline of all organ systems. Mammalian somatic cells in culture display a limited proliferative life span, at the end of which they undergo an irreversible cell cycle arrest known as replicative senescence. Whether cellular senescence contributes to organismal aging has been controversial. We investigated telomere dysfunction, a recently discovered biomarker of cellular senescence, and found that the number of senescent fibroblasts increases exponentially in the skin of aging baboons, reaching >15% of all cells in very old individuals. In addition, the same cells contain activated ataxia-telangiectasia mutated kinase and heterochromatinized nuclei, confirming their senescent status.

Aging↗
PubMed2000

(TRANS)GENE SILENCING IN PLANTS: How Many Mechanisms?

Epigenetic silencing of transgenes and endogenous genes can occur at the transcriptional level (TGS) or at the posttranscriptional level (PTGS). Because they can be induced by transgenes and viruses, TGS and PTGS probably reflect alternative (although not exclusive) responses to two important stress factors that the plant's genome has to face: the stable integration of additional DNA into chromosomes and the extrachromosomal replication of a viral genome. TGS, which results from the impairment of transcription initiation through methylation and/or chromatin condensation, could derive from the mechanisms by which transposed copies of mobile elements and T-DNA insertions are tamed. PTGS, which results from the degradation of mRNA when aberrant sense, antisense, or double-stranded forms of RNA are produced, could derive from the process of recovery by which cells eliminate pathogens (RNA viruses) or their undesirable products (RNA encoded by DNA viruses). Mechanisms involving DNA-DNA, DNA-RNA, or RNA-RNA interactions are discussed to explain the various pathways for triggering (trans)gene silencing in plants.

Journal Article↗
PubMed2003

Double-stranded RNA activates a p38 MAPK-dependent cell survival program in biliary epithelia.

Double-stranded RNA (dsRNA) is produced during replicative viral infection or genotoxic stress. Thus knowledge of the cellular response to dsRNA is necessary to understand the effects of DNA damage or viral infection in biliary epithelia. We assessed the effect of dsRNA on biliary epithelial cell proliferation and apoptosis and the role of the stress-activated p38 MAPK signaling pathway in these responses. dsRNA did not induce apoptosis or proliferation in Mz-ChA-1 human malignant cholangiocytes, but decreased cytotoxicity induced by camptothecin or tumor necrosis factor-related apoptosis inducing ligand and decreased activity of caspases 3, 8, and 9. Furthermore, dsRNA increased p38 MAPK and JNK kinase active site phosphorylation but had no effect on either MAPK kinase (MEK)1/2 or protein kinase R phosphorylation. Inhibition of p38 MAPK with SB-203580 increased basal caspase activity. Thus dsRNA stimulates a p38 MAPK-dependent cell-survival pathway in biliary epithelial cells that may modulate the response of the biliary epithelia to dsRNA produced during genotoxic injury or virus infection.

Apoptosis↗
PubMed2003

Common fragile sites.

Aphidicolin-induced common fragile sites are site-specific gaps or breaks seen on metaphase chromosomes after partial inhibition of DNA synthesis. These fragile sites were first recognized during the early studies of the fragile X syndrome and are induced by the same conditions of folate or thymidylate stress used to induce the fragile X site. Common fragile sites are normally stable in cultured human cells. However, following induction with replication inhibitors, they display a number of characteristics of unstable and highly recombinogenic DNA. From the many studies that have cloned and characterized fragile sites, it is now known that these sites extend over large regions, are associated with genes, exhibit late or delayed replication, and contain regions of high flexibility but are otherwise unremarkable in sequence. Studies showing that fragile sites and their associated genes are frequently deleted or rearranged in cancer cells have clearly demonstrated their importance in genome instability in tumorigenesis. Yet until recently, very little was known about the molecular mechanisms involved in their stability. Recent findings showing that the key checkpoint genes ATR and BRCA1 are critical for genome stability at fragile sites have shed new light on these mechanisms and on the biological significance of common fragile sites.

Animals↗
PubMed2004

The functional architecture of the nucleus as analysed by ultrastructural cytochemistry.

Ultrastructural cytochemistry has been, for many years now, a major tool for investigating structure-function relationships in the cell nucleus. It has been essential in approaching the roles which different nuclear structural constituents can play in nuclear functions. This article briefly summarises transmission electron microscopic studies aimed at characterising in situ nuclear architectural domains and their involvement in main nuclear functions, such as DNA replication, hnRNA transcription and pre-mRNA processing. It discusses the importance of ultrastructural cytochemistry in high resolution analyses of intranuclear distribution of chromatin domains and their topological relationships with other structural interphase nuclear constituents. It puts forward the central role of the perichromatin region as a functional nuclear domain. Finally, it attempts to critically evaluate some future applications of ultrastructural investigations of the nucleus and stresses the importance of combining them with light microscopic analyses of living cells.

Animals↗
PubMed2005

Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival.

Mcl1p is an essential fission yeast chromatin-binding protein that belongs to a family of highly conserved eukaryotic proteins important for sister chromatid cohesion. The essential function is believed to result from its role as a Pol1p (polymerase alpha) accessory protein, a conclusion based primarily on analogy to Ctf4p's interaction with Pol1p. In this study, we show that Mcl1p also binds to Pol1p with high affinity for the N terminus of Pol1p during S phase and DNA damage. Characterization of an inducible allele of mcl1+, (nmt41)mcl1-MH, shows that altered expression levels of Mcl1p lead to sensitivity to DNA-damaging agents and synthetic lethality with the replication checkpoint mutations rad3Delta, rqh1Delta, and hsk1-1312. Further, we find that the overexpression of the S-phase checkpoint kinase, Cds1, or the loss of Hsk1 kinase activity can disrupt Mcl1p's interaction with chromatin and Pol1p during replication arrest with hydroxyurea. We take these data to mean that Mcl1p is a dynamic component of the polymerase alpha complex during replication and is important for the replication stress response in fission yeast.

Alleles↗
PubMed1995

Effect of dietary antioxidants on dieldrin-induced hepatotoxicity in mice.

An increasing number of reports suggest that oxidative stress plays a role in the toxicity of various xenobiotics, including organochlorine pesticides and drugs such as phenobarbital. Antioxidants appear to be protective against the damage induced by an acute dose of endrin, supporting the theory of a role for reactive oxygen in the toxicity of this class of compounds. The current study examined the effects of the dietary administration of vitamin C (400 mg/kg diet) or vitamin E (200 mg DL-alpha-tocopherol acetate/kg diet) on hepatotoxicity induced by subchronic (7 or 28 days) feeding of dieldrin (1, 3 and 10 mg/kg diet) to male B6C3F1 mice. Hepatoxicity induced by feeding of dieldrin for 28 days was evidenced by liver enlargement, hypertrophy of centrolobular hepatocytes, induction of hepatic ethoxyresorufin O-deethylase activity, and increased DNA synthesis in hepatocytes, particularly in centrolobular hepatocytes. Neither vitamin inhibited the dose-dependent increase in liver/body weight ratios, hypertrophy of centrolobular hepatocytes, or induction of hepatic ethoxyresorufin O-deethylase. Vitamin E, however, inhibited hepatic DNA synthesis at all dietary intakes of dieldrin, while vitamin C was inhibitory at 1 and 3, but stimulatory at 10 mg dieldrin per kg diet. The major changes in DNA labeling occurred in the centrolobular zones, but were not consistently inhibited by vitamins C or E. The ability of antioxidant vitamins to inhibit dieldrin-induced hepatic DNA synthesis suggests oxidative stress is involved in the toxicity of this compound; however, the inability of these vitamins to prevent all hepatotoxic changes indicates other factors are also involved.

Animals↗
PubMed2005

Activation of p38 and N-acetylcysteine-sensitive c-Jun NH2-terminal kinase signaling cascades is required for induction of apoptosis in Parkinson's disease cybrids.

Cytoplasmic hybrid cells (cybrids) are created by selective amplification of mitochondrial genes against constant nuclear genetic and environmental backgrounds. Cybrids from patients with sporadic Parkinson's disease (PD) recapitulate disease features such as decreased complex I activity, increased oxidative stress, elevated activation of NF-kappaB, and production of Lewy body inclusions. We examined the activation of signaling pathways and NF-kappaB in PD cybrids after exposure to MAPK inhibitors and/or the antioxidant N-acetylcysteine (NAC). Under basal replicating conditions, PD cybrids have decreased viability that is associated with increased DNA condensation and poly-ADP ribose polymerase (PARP) cleavage as well as elevated p38 and JNK activity. Pharmacological inhibition of oxidative stress diminished the elevated p38, JNK activity and PARP cleavage, and enhanced PD cybrid viability. PD mitochondrial genes expressed in cybrids stimulate pro-apoptotic cell signaling and biochemistry through oxidative stress. These results support development of antioxidative therapeutics for PD.

Acetylcysteine↗
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↗
PubMed1987

Yeast regulatory sequences preferentially adopt a non-B conformation in supercoiled DNA.

Mung bean nuclease was used to probe for DNA unwinding in torsionally-stressed chimeric plasmids containing two micron plasmid sequences and the yeast LEU2 gene in a pBR322 vector. The yeast sequences are cleaved at only two sites, both of which map to regulatory regions: (1) the autonomously replicating sequence (ARS), an origin of DNA replication, of the two micron plasmid and (2) the transcription terminator region of the LEU2 gene. Nucleotide level analysis of the nuclease cleavage pattern shows that an A + T-rich structure, distinct from other non-B DNA conformations, is recognized. A computer analysis reveals that A + T content alone is not sufficient to explain the preferential occurrence of the A + T-rich structure in the ARS over other sequences of equal A + T content. The A + T-rich structure detected in the ARS maps to sequences required for DNA replication. Our findings demonstrate the DNA conformational flexibility of certain yeast regulatory regions and provide support for the hypothesis that the A + T-rich sequence in the ARS plays a role in DNA unwinding during the initiation of DNA replication.

Base Composition↗
PubMed2001

DNA polymerases II and V mediate respectively mutagenic (-2 frameshift) and error-free bypass of a single N-2-acetylaminofluorene adduct.

The NarI sequence represents a strong mutation hot spot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF), a strong chemical carcinogen. Only when bound to the third (underlined) guanine (5'-GGCGCC-->GGCC) can AAF trigger frameshift mutations, suggesting the involvement of a slipped replication intermediate with a two-nucleotide bulge. While base substitutions induced by UV light or abasic sites require DNA polymerase V (Pol V; umuDC), the AAF-induced -2 frameshift pathway requires DNA polymerase II, the polB gene product. Interestingly, error-free bypass of the G-AAF adduct requires Pol V. The genes encoding both Pol II and Pol V are induced by the SOS regulon, a co-ordinated cellular response to environmental stress. A given lesion, G-AAF, can thus be bypassed by two SOS-controlled DNA polymerases (II and V), generating mutagenic (-2 frameshifts) and error-free replication products respectively. Therefore both Pol II and Pol V can compete for the blocked replication intermediate in the vicinity of the lesion and engage in replication by transiently replacing the replicative DNA Pol III. Our data suggest that, in order to cope with the large diversity of existing DNA lesions, cells use a single or a combination of translesional DNA polymerases to achieve translesion synthesis.

2-Acetylaminofluorene↗
PubMed2003

Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver.

After several weeks of treatment, levels of alanine aminotransferase (ALT) increase in 50% of patients treated with tacrine for Alzheimer's disease. We looked for progressive effects on DNA to explain delayed toxicity. We first studied the in vitro effects of tacrine on DNA replication and topoisomerase-mediated DNA relaxation. We then treated mice with doses of tacrine reproducing the human daily dose on a body area basis and studied the effects of tacrine administration for up to 28 days on hepatic DNA, mitochondrial function, and cell death. In vitro, tacrine impaired DNA polymerase gamma-mediated DNA replication and also poisoned topoisomerases I and II to increase the relaxation of a supercoiled plasmid. In vivo, administration of tacrine markedly decreased incorporation of [(3)H]thymidine into mitochondrial DNA (mtDNA), progressively and severely depleted mtDNA, and partly unwound supercoiled mtDNA into circular mtDNA. Incorporation of [(3)H]thymidine into nuclear DNA (nDNA) was barely decreased, and nDNA levels were unchanged. After 12 to 28 days of treatment, administration of tacrine increased p53, Bax, mitochondrial permeability transition, cytosolic cytochrome c, and caspase-3 activity and triggered hepatocyte apoptosis and/or necrosis. In conclusion, the intercalating drug tacrine poisons topoisomerases and impairs DNA synthesis. Tacrine has been shown to accumulate within mitochondria, and it particularly targets mtDNA. After several weeks of treatment, the combination of severe mtDNA depletion and a genotoxic stress enhancing p53, Bax, and permeability transition trigger hepatocyte necrosis and/or apoptosis.

Animals↗