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PubMed2002

An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates.

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.

Animals↗

PubMed2003

Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells.

Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16(INK4a)-associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16(INK4a) and milder upregulation of p53 and p21(WAF1), which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4-overexpressing cells remain intact.

Adult↗

PubMed1995

Stimulation of DNA synthesis by mouse DNA helicase B in a DNA replication system containing eukaryotic replication origins.

A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far. We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin. In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin. We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen. DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase. DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A. A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B. Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system.

Adenosine Triphosphatases↗

PubMed1989

Relationship between organization of the actin cytoskeleton and the cell cycle in normal and adenovirus-infected rat cells.

Flow cytometry and staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin were used to investigate organization of the actin cytoskeleton in rat embryo cells at different stages of normal and adenovirus E1A-induced cell cycles. In uninfected cells in G0-G1 and S phases, actin was predominantly in the form of stress fibers. In G2, this organization changed to peripheral rings of thin filaments, while during mitosis, actin had a diffuse distribution. Infection of quiescent rat cells by adenovirus caused them to enter the cell cycle and replicate DNA and also caused disruption of stress fibers. Rapid disappearance of stress fibers and the appearance of peripheral rings of actin filaments began from 13 h after infection and closely followed synthesis of the E1A proteins. Infected cells began S phase at about 24 h after infection, and cells in G2 and mitosis were seen from 30 to 50 h. Thus, disruption of the actin cytoskeleton is an early effect of E1A and not an indirect consequence of the entry of infected cells into the cell cycle.

Actins↗

PubMed2001

Threonine-11, phosphorylated by Rad3 and atm in vitro, is required for activation of fission yeast checkpoint kinase Cds1.

Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T(11)Q(12). Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1(-) null mutant. Cds1(T11A) was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.

Adenosine Triphosphatases↗

PubMed1994

Aldehyde-induced protein-DNA crosslinks disrupt specific stages of SV40 DNA replication.

Aldehydes with specific protein-DNA crosslinking ability disrupted simian virus 40 (SV40) DNA replication to cause replication fork failure by the 40S intermediate pathway, in which replicating viral genomes become inactivated and torsionally stressed. In contrast, aldehydes without detectable protein-DNA crosslinking ability had no effect on SV40 DNA replication during the 10 min exposure times employed. This indicates that protein-DNA crosslinks block either DNA polymerase or the entire replication complex. Replication failure by the 40S pathway is known to initiate recombinational events in the damaged SV40 replicons. Similar events in cellular replicons may play a role in the clastogenic effects of formaldehyde. In addition, formaldehyde and acrolein caused accumulation of catenated (topologically linked) SV40 daughter chromosomes--a signature of topoisomerase II inhibition.

Acrolein↗

PubMed1998

Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: implications for the mechanism of fragile site induction.

The FRA3B at 3p14.2 is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin or folate stress. The molecular basis for chromosome fragility at FRA3B is unknown. In contrast to the rare fragile sites, including FRAXA, no repeat motifs, such as trinucleotide repeats, have been identified within FRA3B. Several lines of evidence suggest that fragile sites are regions of DNA whose replication is unusually sensitive to interference. We have used fluorescence in situ hybridization to determine the relative timing of replication of FRA3B sequences. Our studies revealed that FRA3B sequences are late replicating. Exposure to aphidicolin, an inhibitor of both DNA polymerase alpha and delta, results in a reproducible delay in the timing of replication, and some cells enter G2without having completed replication of FRA3B sequences. Our results support a model in which common fragile sites are sequences that initiate replication late in S phase or are slow to replicate, and the chromosomal breaks and gaps observed in metaphase cells are due to unreplicated DNA.

Aphidicolin↗

PubMed2025

Regulation of replication timing in Saccharomyces cerevisiae.

In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

Saccharomyces cerevisiae↗

PubMed1983

Cyclophosphamide-induced in vivo sister chromatid exchange in Mus Musculus. II: Effect of age and genotype on sister chromatid exchange, micronuclei and metaphase index.

In vivo cyclophosphamide-induced sister chromatid exchanges (SCEs) micronuclei, and metaphase indices were assessed in two age groups (10.8 +/- 0.9 weeks' an 33.1 +/- 1.3 weeks' old) of female mice from three genetic strains (C3H/S, C57BL/6J, and Balb/c). In general, older animals showed diminished SCE induction over their younger counterparts. The relative difference between individuals of the two ages is strain-dependent. Unlike C57BL/6J and Balb/c, strain C3H/S showed significantly lower SCE values in the older animals at every cyclophosphamide treatment. It may reflect on the possible involvement of genetic determinant(s) for the component(s) of SCE formation during aging. Frequencies of micronuclei, however, were consistently higher in older animals than in their younger counterparts. Furthermore, cytotoxicity of cyclophosphamide, as reflected in metaphase indices, was also higher in older animals. Lower metaphase indices associated with higher micronuclei levels in older individuals may suggest a decline in the rate of cellular replication in these animals. Furthermore, the lower metaphase indices associated with lower SCE values, and increasing micronuclei levels accompanied by decreasing SCE frequencies in older animals, may reflect reduced DNA repair ability during aging. These results support the hypothesis of genotype-dependent decline in the rate of DNA repair and replication during aging, particularly under stressed conditions.

Aging↗

PubMed2006

Reactivation from quiescence does not coincide with a global induction of herpes simplex virus type 1 transactivators.

Herpes simplex virus type 1 (HSV-1) reactivates from a small fraction of latently infected neurons in vivo and neuronally differentiated (ND), quiescently infected (QIF)-PC12 cells in vitro. This may be the result of reactivation initiating in only a few cells, or reactivation followed by premature termination of the productive virus life cycle in many or even a majority of cells. To examine the viral stress response, HSV-1 promoters of representative alpha, beta, and gamma class genes were examined in ND- and QIF-PC12 cells after treatments with agents known to induce reactivation. HSV-1 promoters displayed variable levels of basal gene expression in ND-PC12 cells ranging from 2 to 1,200 times the level of the control vector pGL3-Basic. Expression of the latency associated transcript (LAT) was greatest, with representatives of the alpha class exhibiting greater expression than the beta and gamma classes. The HSV-1 promoters examined did not respond dramatically to stress treatments. The viral gene response was also measured during the initiation of reactivation of a cryptic HSV-1 genome after forskolin treatment, under conditions that restricted DNA replication. During the first 24 h after stress induction the response was limited. By 48 h post-forskolin treatment, only modest increases occurred for ICP0, ICP4, and LAT transcripts, reaching levels of no greater than 2.2 times mock treated levels. In contrast, ICP27, ribonucleotide reductase (RR), and VP16 promoters did not respond. These findings indicate that reactivation from QIF-PC12 cells does not result in a global response of the specific HSV-1 genes tested, when assessed at the population level. These data support the hypothesis that stress-induced reactivation initiates in a minority of cells.

Animals↗

PubMed2006

Tipin and Timeless form a mutually protective complex required for genotoxic stress resistance and checkpoint function.

Tipin is a mammalian protein that interacts with Timeless, which plays a role in DNA damage checkpoint responses. Here, we show that Tipin is a nuclear protein that associates with the replicative helicase and protects cells against genotoxic agents. Tipin is required for efficient cell cycle arrest in response to DNA damage, and depletion of Tipin renders cells sensitive to ionizing radiation as well as replication stress. Loss of Tipin results in spontaneous gamma-H2AX foci, a marker for DNA double-strand breaks. We find that Tipin and Timeless form a complex that maintains the level of both proteins in cells and that the loss of either one will lead to the loss of the interacting partner. This observation explains the similar checkpoint phenotypes observed in both Tipin- and Timeless-depleted cells.

Carrier Proteins↗

PubMedDate not supplied

Mitochondrial DNA replication, nucleoside reverse-transcriptase inhibitors, and AIDS cardiomyopathy.

Nucleoside reverse-transcriptase inhibitors (NRTIs) in combination with other antiretrovirals (HAART) are the cornerstones of current AIDS therapy, but extensive use brought mitochondrial side effects to light. Clinical experience, pharmacological, cell, and molecular biological evidence links altered mitochondrial (mt-) DNA replication to the toxicity of NRTIs in many tissues, and conversely, mtDNA replication defects and mtDNA depletion in target tissues are observed. Organ-specific pathological changes or diverse systemic effects result from and are frequently attributed to HAART in which NRTIs are included. The shared features of mtDNA depletion and energy depletion became key observations and related the clinical and in vivo experimental findings to inhibition of mtDNA replication by NRTI triphosphates in vitro. Subsequent to those findings, other observations suggested that mitochondrial energy deprivation is concomitant with or the result of mitochondrial oxidative stress in AIDS (from HIV, for example) or from NRTI therapy itself.

Acquired Immunodeficiency Syndrome↗

PubMed2004

Mitochondrial oxidative stress in human hepatoma cells exposed to stavudine.

The toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is linked to altered mitochondrial DNA (mtDNA) replication and subsequent disruption of cellular energetics. This manifests clinically as elevated concentrations of lactate in plasma. The mechanism(s) underlying how the changes in mtDNA replication lead to lactic acidosis remains unclear. It is hypothesized that mitochondrial oxidative stress links the changes in mtDNA replication to mitochondrial dysfunction and ensuing NRTIs toxicity. To test this hypothesis, changes in mitochondrial function, mtDNA amplification efficiency, and oxidative stress were assessed in HepG2-cultured human hepatoblasts treated with the NRTI stavudine (2',3'-didehydro-2',3'-deoxythymidine or d4T) for 48 h. d4T produced significant mitochondrial dysfunction with a 1.5-fold increase in cellular lactate to pyruvate ratios. In addition, d4T caused a dose-dependent decrease in mtDNA amplification and a correlative increase in abundance of markers of mitochondrial oxidative stress. Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, MnTBAP, a catalytic antioxidant, ameliorated or reversed d4T-induced changes in cell injury, energetics, mtDNA amplification, and mitochondrial oxidative stress. In conclusion, d4T treatment elevates mitochondrial reactive oxygen species (ROS), enhances mitochondrial oxidative stress, and contributes mechanistically to NRTI-induced toxicity. These deleterious events may be potentiated in acquired immunodeficiency syndrome (AIDS) by human immunodeficiency virus (HIV) infection itself, coinfection (e.g., viral hepatitis), aging, substance, and alcohol use.

Carcinoma, Hepatocellular↗

PubMed2003

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts.

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.

Ataxia Telangiectasia↗

PubMed2000

Role of oxidative stress in telomere length regulation and replicative senescence.

Replicative senescence is tied into organismal aging processes in more than one respect, and telomeres appear to be the major trigger of replicative senescence under many conditions in vitro and in vivo. However, the structure-function relationships in telomeres, the mechanisms of telomere shortening with advancing replicative age, and the regulation of senescence by telomeres are far from understood. Combining recent data on telomere structure, function of telomere-binding proteins, and sensitivity of telomeres to oxidative damage, an integrative model of telomere shortening and signaling is developed. The model suggests that t-loop formation hinders access of repair proteins to telomeres, leading to accumulation of a basic sites and single-strand breaks. These might contribute to accelerated telomere shortening by transient stalling of replication as well as, if present in high concentrations, to a relief of torsional tension which might destabilize the telomeric loop structure. As a result, the single-stranded G-rich overhang, which is present at the very ends of telomeres but is normally protected at the base of the telomeric loop, will be exposed to the nucleoplasm. Free G-rich telomeric single strands are a strong inductor of the p53 pathway, and exposure of the overhangs seems to be the first step in the signal transduction cascade to replicative senescence.

Animals↗

PubMed1998

The structure of supercoiled intermediates in DNA replication.

We studied the structure of replication intermediates accumulated by Tus-induced arrest of plasmid DNA replication at termination sites. For intermediates generated both in vitro with purified components and in vivo, superhelical stress is distributed throughout the entire partially replicated molecule; daughter DNA segments are wound around each other, and the unreplicated region is supercoiled. Thus, unlinking of parental DNA strands by topoisomerases can be carried out both behind and in front of the replication fork. We explain why previous studies with prokaryotic and eukaryotic replication intermediates discerned only supercoiling in the unreplicated portion.

DNA Replication↗

PubMed2000

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells.

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.

Blotting, Northern↗

PubMedDate not supplied

[The effect of surgical stress on DNA synthesis in liver and brain cells].

Distinct alterations in the rate of DNA synthesis (an increase in the rate of reparation and a decrease in the rate of replication in nuclei and mitochondria) were detected in liver and brain cells during the stress caused by surgical operation. Within 8-10 hrs after the operation the rate of DNA reparation was increased by 40-50% in nuclei of liver cells and by 31-35% in brain cell nuclei. Replication of nuclear DNA was decreased immediately after the operation--by 33% in liver cells and by 50% in brain cells. Within a day after the operation the rate of replication was restored up to the control level in liver cells, while it was still decreased by 30% in brain cells. The rate of mitochondrial DNA synthesis was slightly decreased within the first 12 hrs after the operation: by 12-14% in liver cells and by 18-20% in brain cells. The stress, and particularly postoperative stress, exhibited pronounced action on structure and various systems of DNA synthesis in cells of various tissues.

Animals↗