Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA sensing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed2003

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae.

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.

Cell Cycle↗
PubMed2006

Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus.

The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Terminal DNA structure and ATP influence binding parameters of the DNA-dependent protein kinase at an early step prior to DNA synapsis.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the non-homologous end-joining pathway of DNA double-strand break repair in mammalian cells. The ability of DNA-PKcs to sense and respond to different terminal DNA structures is postulated to be important for its regulatory function. It is unclear whether discrimination occurs at the time of formation of the initial protein-DNA complex or later, at the time of formation of a paired, or synaptic complex between opposing DNA ends. To gain further insight into the mechanism of regulation, we characterized the binding of DNA-PKcs to immobilized DNA fragments that cannot undergo synapsis. Results showed that DNA-PKcs strongly discriminates between different terminal structures at the time of initial complex formation. Although Ku protein stabilizes DNA-PKcs binding overall, it is not required for discrimination between terminal structures. Base mispairing, temperature and the presence of an interstrand linkage influence the stability of the initial complex in a manner that suggests a requirement for DNA unwinding, reminiscent of the 'open complex' model of RNA polymerase-promoter DNA interaction. ATP and a nonhydrolyzable ATP analog also influence the stability of the DNA-PKcs*DNA complex, apparently by an allosteric mechanism that does not require DNA-PKcs autophosphorylation.

Adenosine Triphosphate↗
PubMed1991

A genetic tool used to identify thioredoxin as a mediator of a growth inhibitory signal.

Loss of sensitivity to growth inhibitory polypeptides is likely to be one of the events that participates in the formation of some tumors and might be caused by inactivation or loss of the genetic elements that transduce these extracellular signals. The isolation of such a gene was achieved by randomly inactivating genes by an anti-sense complementary DNA expression library followed by direct selection for growth in the presence of an inhibitory polypeptide. Thus, a gene whose inactivation conveyed growth resistance to interferon-gamma (IFN-gamma) was isolated. Sequence analysis showed complete identity with human thioredoxin, a dithiol reducing agent, implicated here in the IFN-gamma-mediated growth arrest of HeLa cells.

Blotting, Northern↗
PubMed1995

Thermoregulation of virB transcription in Shigella flexneri by sensing of changes in local DNA superhelicity.

Transcription of the virB gene, a transcriptional regulator of invasion genes on the large plasmid of Shigella flexneri, is strictly regulated by growth temperature; when bacteria are grown at 37 degrees C, virB transcription is highly activated, while at 30 degrees C the level of virB transcription decreases to less than 5% of that at 37 degrees C. Transcription from the virB promoter is activated by VirF, which is encoded on the same plasmid, in a DNA superhelicity-dependent manner (T. Tobe, M. Yoshikawa, T. Mizuno, and C. Sasakawa, J. Bacteriol. 175:6142-6149, 1993). Here we provide evidence supporting the involvement of negative superhelicity in the thermoregulation of virB transcription. A local negatively supercoiled domain in the virB promoter region was created by activating a divergent transcription from the T7 RNA polymerase-dependent promoter, phi 10, which was placed upstream of the virB promoter in the opposite orientation. Transcription from the virB promoter was activated even at 30 degrees C by induction of divergent transcription. Levels of virB transcription correlated with levels of expressed T7 RNA polymerase. Transcriptional activation of virB by the system depended completely upon VirF function. The level of virB transcription achieved by introducing a negatively supercoiled domain was enough to give rise to expression of invasion capacity at 30 degrees C. These results indicated that the repression of virB transcription at 30 degrees C was caused by a reduction in negative superhelicity around the virB promoter region at 30 degrees C.

Bacterial Outer Membrane Proteins↗
PubMed2004

Biosynthesis and accumulation of sterols.

In recent years, the impressive development of molecular genetics tools, the sequencing of the Arabidopsis thaliana genome, the availability of DNA or transposon tagged mutants, and the multiple possibilities offered by stable transformation with DNA in sense and antisense orientation have enabled the application of a strategy of gain or loss of function to study the sterol biosynthesis pathway. Here we describe the results obtained with these techniques. The results essentially confirm data obtained previously with sterol biosynthesis inhibitors (SBIs) and enable the precise dissection of biosynthetic pathways. We discuss the advantages and disadvantages of molecular genetics techniques as applied to sterol metabolism. The greater selectivity of these techniques constitutes an invaluable advantage and has led to the discovery of a role for sterols in plant development.

Amino Acid Sequence↗
PubMed2006

Human lymphoblastoid proteome analysis reveals a role for the inhibitor of acetyltransferases complex in DNA double-strand break response.

A DNA double-strand break (DSB) is highly cytotoxic; it emerges as the type of DNA damage that most severely affects the genomic integrity of the cell. It is essential that DNA DSBs are recognized and repaired efficiently, in particular, prior to mitosis, to prevent genomic instability and eventually, the development of cancer. To assess the pathways that are induced on DNA DSBs, 14 human lymphoblastoid cell lines were challenged with bleomycin for 30 and 240 minutes to establish the fast and more prolonged response, respectively. The proteomes of 14 lymphoblastoid cell lines were investigated to account for the variation among individuals. The primary DNA DSB response was expected to occur within the nucleus; therefore, the nuclear extracts were considered. Differential analysis was done using two-dimensional difference in gel electrophoresis; paired ANOVA statistics were used to recognize significant changes in time. Many proteins whose nuclear levels changed statistically significantly showed a fast response, i.e., within 30 minutes after bleomycin challenge. A significant number of these proteins could be assigned to known DNA DSB response processes, such as sensing DSBs (Ku70), DNA repair through effectors (high-mobility group protein 1), or cell cycle arrest at the G(2)-M phase checkpoint (14-3-3 zeta). Interestingly, the nuclear levels of all three proteins in the INHAT complex were reduced after 30 minutes of bleomycin challenge, suggesting that this complex may have a role in changing the chromatin structure, allowing the DNA repair enzymes to gain access to the DNA lesions.

Amino Acid Sequence↗
PubMed2026

Context-Dependent cGAS-STING Activation Shapes Metastatic Progression and Dormancy.

Cancer cells survive, proliferate, and metastasize in part because the immune system fails to detect and eliminate them. Moreover, the tumor microenvironment (TME) that surrounds the tumor supports cancer cell survival and resistance to chemo- and immunotherapies by inhibiting antitumor immune responses and thereby reducing the efficacy of immunotherapeutic interventions. cGAS-STING signaling senses cytoplasmic DNA and coordinates innate immune responses that shape tumor-intrinsic outcomes and the TME. Emerging evidence reveals a context-dependent, dualistic role for cGAS-STING in metastatic progression and cancer dormancy. Acute, robust activation in antigen-presenting cells promotes type I interferon responses, leading to suppression of tumor growth. By contrast, chronic, low-level cancer-intrinsic STING signaling can engage inflammatory programs that foster immune suppression and therapy resistance. Dormant disseminated tumor cells exploit niche cues to downregulate STING signaling and evade immune detection, whereas reactivation of dormant cells often involves restoration of STING activity that can promote immune elimination. In this article, we review mechanisms linking genome instability and cytoplasmic DNA to STING activation, summarize evidence for tumor-suppressive versus tumor-promoting functions across metastatic niches, and discuss how STING agonists and combination strategies may be optimized to maximize antitumor immunity while avoiding protumorigenic effects.

Humans↗
PubMed1991

Sensitivity and specificity of a universal primer set for the rapid diagnosis of dengue virus infections by polymerase chain reaction and nucleic acid hybridization.

A set of sense and anti-sense oligomeric DNA primers, degenerate in the third "wobble" base position of codons so as to match all known dengue virus sequences, was evaluated as universal primers in a polymerase chain reaction (PCR) assay for the rapid diagnosis of dengue virus infections. Virus-specific complementary DNA (cDNA) was prepared by reverse transcription (RT) of total RNA extracted from serum. Amplified cDNA was identified by nucleic acid hybridization with four serotype-specific, oligomeric DNA probes. Using sera from patients admitted with dengue fever, RT/PCR followed by nucleic acid hybridization using radiolabeled probes was 68% sensitive (50/74; 95% confidence interval [CI] = 57-78%) and 100% specific. Chemiluminescent detection of hybridized products was 62% sensitive (26/42; 95% CI = 46-75%). Using specimens from which a virus isolate had been obtained, RT/PCR followed by nucleic acid hybridization with radiolabeled probes was 80% sensitive (40/50; 95% CI = 69-91%) and 100% specific. The results suggest that RT/PCR using degenerate primers is a sensitive and specific method for the detection of dengue viruses in clinical specimens.

DNA Probes↗
PubMed2000

A role for the p38 mitogen-acitvated protein kinase pathway in the transcriptional activation of p53 on genotoxic stress by chemotherapeutic agents.

The tumor suppressor p53 plays a central role in sensing damaged DNA and orchestrating the consequent cellular responses. However, how DNA damage leads to the activation of p53 is still poorly understood. In this study, we have found that the p38 mitogen-activated protein kinase (MAPK) plays a key role in the activation of p53 by genotoxic stress when provoked by chemotherapeutic agents. Indeed, we found that blockade of p38 prevents stimulation of the transcriptional activity of p53 and that activation of the p38 pathway is sufficient to stimulate p53 function. Furthermore, we observed that p38 does not affect the accumulation of p53 in response to DNA damage or its nuclear localization. In contrast, we observed that p38 associates physically with p53, and we provide evidence that this MAPK phosphorylates the NH2-terminal transactivation domain of p53 in serine 33, thereby stimulating its functional activity. Moreover, inhibition of the p38 MAPK diminished the apoptotic fraction of cells exposed to chemotherapeutic agents and increased cell survival, thus suggesting a role for p38 activation in the apoptotic response to genotoxic stress when elicited by drugs used in cancer therapy.

3T3 Cells↗
PubMed1990

Detection of interleukin-5 messenger RNA in Reed-Sternberg cells of Hodgkin's disease with eosinophilia.

Hodgkin's disease of nodular sclerosis and mixed cellularity subtypes is frequently associated with eosinophilia. To determine if interleukin-5 (IL-5) is implicated in producing the eosinophilia, we performed in situ hybridization studies on cytopreparations of 16 cases of Hodgkin's disease with eosinophilia as well as cells from various controls. A single-stranded, anti-sense complementary DNA (cDNA) probe coding for a portion of the human IL-5 molecule was tail-labeled with digoxigenin -11-dUTP using terminal transferase, and then hybridized to messenger RNA (mRNA) within cells. An alkaline-phosphatase-conjugated antibody directed to digoxigenin was used with a chromogenic substrate to detect hybridized probe within cells. In all seven cases of nodular sclerosis subtype and nine cases of mixed cellularity subtype with eosinophilia, there was strong hybridization signal localizable to the cytoplasm of morphologically identifiable Reed-Sternberg cells and variants. Similar activity was detected only in rare cells from three normal spleens, and was undetectable in two cell lines used as a negative control and in one case of Hodgkin's disease without eosinophilia. Pretreatment of the cytopreparations with the RNase inhibitor diethylpyrocarbonate greatly increased the hybridization signal. Based on this controlled study, we conclude that mRNA coding for IL-5 is expressed in Reed-Sternberg cells and variants. This observation may explain the eosinophilia associated with Hodgkin's disease and provide insight into the origin of the Reed-Sternberg cell.

Eosinophilia↗
PubMed1993

A codon 248 p53 mutation retains tumor suppressor function as shown by enhancement of tumor growth by antisense p53.

Codon 248 in domain iv of the highly conserved region of the p53 gene is a frequent site of mutations associated with sporadic cancers and the familial cancer syndrome (Li-Fraumeni syndrome). Therefore, a characterization of the functional significance of a codon 248 mutation is of interest. We used antisense RNA methodology to study the role of the wild-type and mutated p53 gene in cell growth and tumorigenesis. We introduced wild-type p53 complementary DNA in sense or antisense orientation under control of a beta-actin promoter into human non-small cell lung cancer cell line H322a which has a codon 248 mutation (G to T) and WTH226b which has wild type p53. The biological properties and p53 expression of stable G418-resistant clones were analyzed. We observed that in both cell lines antisense RNA expression significantly reduced p53 mRNA and protein production; it also caused increases in growth rate in cell cultures and in tumorigenicity in nu/nu mice for both cell types, suggesting that the mechanism by which p53 suppresses cell proliferation and tumorigenesis is not always abrogated by a codon 248 mutation.

Animals↗
PubMed1996

Fiber-optic fluorometric sensing of polymerase chain reaction-amplified DNA using an immobilized DNA capture protein.

A fiber-optic assay for amplified DNA products has been developed. Modifications of the DNA capture strategy described previously by Kemp et al. [Proc. Natl. Acad. Sci. USA 86, 2423-2427 (1989)] were made that allowed selective binding of DNA labeled during the amplification process to the sensing surface of fused silica fibers. The gene for a chimeric protein composed of the IgG-binding beta 2 subdomain of streptococcal protein G fused with the DNA binding domain of yeast GCN4 was constructed, and this PG/GCN4 protein was overexpressed in Escherichia coli. The purified protein was noncovalently bound to IgG-modified fibers utilizing strong and specific interactions between the protein G beta 2 domain and goat IgG that had been covalently immobilized on the fiber surface. Nanomolar concentrations of amplified DNA labeled with the fluorophore tetramethylrhodamine and the AP-1 consensus nucleotide sequence recognized by GCN4 (5'-ATGACTCAT) were rapidly and selectively bound within the evanescent zone of multimode laser-illuminated fibers. Signal from unincorporated fluorescent PCR primer was negligible. Individual fibers could be used for multiple sequential assays, since the fluorescent double-stranded DNA was rapidly and completely stripped from their surfaces with high salt solutions, leaving the IgG-PG/GCN4 DNA binding complex intact to accept another PCR sample.

Amino Acid Sequence↗
PubMed2006

Towards biochips using microstructured optical fiber sensors.

In this paper we present the first incorporation of a microstructured optical fiber (MOF) into biochip applications. A 16-mm-long piece of MOF is incorporated into an optic-fluidic coupler chip, which is fabricated in PMMA polymer using a CO(2) laser. The developed chip configuration allows the continuous control of liquid flow through the MOF and simultaneous optical characterization. While integrated in the chip, the MOF is functionalized towards the capture of a specific single-stranded DNA string by immobilizing a sensing layer on the microstructured internal surfaces of the fiber. The sensing layer contains the DNA string complementary to the target DNA sequence and thus operates through the highly selective DNA hybridization process. Optical detection of the captured DNA was carried out using the evanescent-wave-sensing principle. Owing to the small size of the chip, the presented technique allows for analysis of sample volumes down to 300 nL and the fabrication of miniaturized portable devices.

Biosensing Techniques↗
PubMed2006

BglII-based panhandle and reverse panhandle PCR approaches increase capability for cloning der(II) and der(other) genomic breakpoint junctions of MLL translocations.

Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful for MLL genomic breakpoint junctions and fusion transcripts because MLL has a large number of partner genes. However, genomic panhandle PCR approaches are impeded when the restriction fragment that contains the breakpoint junction is too large to amplify. We devised new panhandle PCR approaches for MLL genomic breakpoint junctions that create the template from BglII restriction fragments by attaching MLL sequence to a BglII site in the partner gene. This leads to the annealing of MLL and its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all from MLL. BglII panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing a BglII overhang and sequence complementary to MLL exon 7 to the 3' ends of BglII digested DNA, and forming the template from the sense strand of DNA. In BglII reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing a BglII overhang and the complement of antisense sequence in MLL exon 10 was ligated to the 3' ends of BglII digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified 5'-MLL-MLLT4-3' and 5'-AFF1-MLL-3' breakpoint junctions. The former is significant because few t(6;11) genomic breakpoint junctions have been sequenced. BglII panhandle PCR approaches increase the possibilities for cloning MLL genomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations.

Adolescent↗
PubMed1991

Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis.

Overexpression of the epidermal growth factor (EGF) receptor (c-erbB) proto-oncogene is a frequent occurrence in human carcinoma and appears to accompany autocrine or paracrine transforming growth factor-alpha expression, which in model systems can result in activation of EGF receptor tyrosine kinase activity and phenotypic transformation. Here we have investigated the transcriptional regulation of the EGF receptor gene, by run-on transcription in isolated nuclei derived from epithelioid tumor lines. The level of transcription was measured at various points on the 100-kilobase pair EGF receptor gene locus, on either sense or antisense DNA strands. We find the level of sense strand transcription along exon 1 is 8-fold higher than transcription in exons 2-26. Primary EGF receptor transcripts appear to pause or terminate prematurely between exons 1 and 2. Termination was mapped to a sequenced region approximately 2 kilobase pairs 3' of exon 1, proximal to a previously reported DNase I hypersensitive site and an enhancer-like activity. Transcription in the CpG-rich region surrounding exon 1 is bidirectional, with antisense transcripts initiating in intron 1 and extending through the coding first exon. Activation of protein kinase C results in a 5-fold induction of EGF receptor transcription, accompanied by a slow release in the block RNA elongation between exon 2 and exon 26, showing that EGF receptor RNA synthesis may be altered by changes in de novo transcription and by a block to RNA elongation.

Amino Acid Sequence↗
PubMed2003

Kinetics of DNA binding with chloroquine phosphate using capacitive sensing method.

The capacitive sensing method has been applied to study the binding of DNA with chloroquine phosphate. DNA was immobilized on a gold electrode surface, self-assembled with thioglycolic acid. The results of a quartz crystal impedance (QCI) study indicate that the reaction of double-strand DNA (dsDNA) with chloroquine includes a fast electrostatic attraction and a slow intercalation of chloroquine into double-strand helix. The real-time experimental data obtained by capacitive sensing also revealed two distinctive kinetics stages during binding of dsDNA with chloroquine, while only one stage exists during reaction of single-strand DNA (ssDNA) with chloroquine. The kinetic parameters were obtained by fitting the real-time experimental data using a two stage reaction model. The rate constants of electrostatic attraction for dsDNA and ssDNA are estimated as 0.014 and 0.018 s(-1), respectively. The rate constant of the second stage of dsDNA is 0.0011 s(-1).

Adsorption↗
PubMedDate not supplied

[Antisense RNA p53 inhibits proliferation of normal and transformed cells].

p53 anti-sense sequences were introduced into normal NIH3T3 and transformed CMS 4 cells by infection with the recombinant retrovirus carrying a repeat of the 5'-terminal fragment of p53 cDNA. Clones selected for G418 resistance showed a marked inhibition of proliferative capacity and a reduced ability to enter DNA replication after stimulation of quiescent cells with serum. Clones showing moderate inhibition of proliferation were shown to contain truncated anti-sense DNA integrated into the genome. The anti-sense DNA was transcribed and it correlated with the reduction of the p53 protein level in the cell clones studied. We conclude that the appropriate expression of p53 appears to be required for cell proliferation.

Animals↗