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Endonuclease
Kinds of Endonuclease Terms modified by Endonuclease Selected AbstractsInnentitelbild: Ultrasensitive and Selective Colorimetric DNA Detection by Nicking Endonuclease Assisted Nanoparticle Amplification (Angew. Chem.ANGEWANDTE CHEMIE, Issue 37 200937/2009) Einen empfindlichen und selektiven kolorimetrischen Nachweis von DNA-Sequenzen beschreiben X.,Liu et,al. in der Zuschrift auf S.,6981,ff. Ihr Verfahren nutzt eine Kombination aus Endonuclease- Nicking-Reaktion und Nanopartikel-Amplifizierung und eignet sich beispielsweise zum Nachweis langer (z.,B. 80-merer) Oligonucleotide mit Einzelbasen-Fehlpaarungs-Selektivität und einer 1000-mal besseren Amplifizierung, ohne dass komplizierte Verfahren angewendet werden müssen, die teure Geräte erfordern. [source] Caspase-dependent and -independent apoptosis of mast cells induced by withdrawal of IL-3 is prevented by Toll-like receptor,4-mediated lipopolysaccharide stimulationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2003Hideshi Yoshikawa Abstract IL-3-dependent mucosal-like mast cells undergo apoptosis upon withdrawal of IL-3. Generally, the apoptosis is mediated by the activation of caspases and inhibited by addition of the pan-caspase inhibitors z-VAD-FMK or BOC-D-FMK. However, DNA fragmentation, a typical characteristic of apoptosis, is not inhibited by z-VAD-FMK or BOC-D-FMK in mast cell apoptosis. In this study, we demonstrate that the apoptosis of mast cells is mediated by both caspase-dependent and -independent mechanisms. The caspase-independent apoptosis is mediated by the translocation of endonuclease,G from mitochondria into nuclei. Withdrawal of IL-3 caused down-regulation of Bcl-xL, resulting in a drop in mitochondrial membrane transition potential followed by the release of cytochrome,c and endonuclease,G from mitochondria. However, stimulation of mast cells through Toll-like receptor,4 (TLR4) by lipopolysaccharide prevented mast cell apoptosis by inducing expression of Bcl-xL. Moreover, the activation of mast cells by LPS is enhanced in the presence of IFN-,, which up-regulates the expression of cell surface TLR4. Taken together, these observations provide evidence that mast cells play importantroles not only in allergic reactions but also in innate immunity recognizing enterobacteria through TLR4, and are regulated differently from allergic inflammation by Th1 cytokines. [source] Polyamines interact with DNA as molecular aggregatesFEBS JOURNAL, Issue 17 2002Luciano D'Agostino New compounds, named nuclear aggregates of polyamines, having a molecular mass of 8000, 4800 and <,1000 Da, were found in the nuclear extracts of several replicating cells. Their molecular structure is based on the formation of ionic bonds between polyamine ammonium and phosphate groups. The production of the 4800 Da compound, resulting from the aggregation of five or more <,1000 Da units, was increased in Caco-2 cells treated with the mitogen gastrin. Dissolving single polyamines in phosphate buffer resulted in the in vitro aggregation of polyamines with the formation of compounds with molecular masses identical to those of natural aggregates. After the interaction of the 4800 Da molecular aggregate with the genomic DNA at 37 °C, both the absorbance of DNA in phosphate buffer and the DNA mobility in agarose gel increased greatly. Furthermore, these compounds were able to protect the genomic DNA from digestion by DNase I, a phosphodiesterasic endonuclease. Our data indicate that the nuclear aggregate of polyamines interacts with DNA phosphate groups and influence, more efficaciously than single polyamines, both the conformation and the protection of the DNA. [source] Solution structure of a zinc-finger domain that binds to poly-ADP-riboseGENES TO CELLS, Issue 2 2010Shin Isogai Poly-ADP-ribosylation is a unique post-translational modification that controls various nuclear events such as repair of DNA single-strand breaks. Recently, the protein containing the poly-ADP-ribose (pADPr)-binding zinc-finger (PBZ) domain was shown to be a novel AP endonuclease and involved in a cell cycle checkpoint. Here, we determined the three-dimensional structure of the PBZ domain from Drosophila melanogaster CG1218-PA using NMR spectroscopy. The domain folds into a C2H2-type zinc-finger structure in an S configuration, containing a characteristic loop between the zinc-coordinating cysteine and histidine residues. This is distinct from the structure of other C2H2-type zinc fingers. NMR signal changes that occur when pADPr binds to the PBZ domains from CG1218-PA and human checkpoint with FHA (forkhead-associated) and ring finger (CHFR) and mutagenesis suggest that a surface relatively well conserved among PBZ domains may serve as a major interface with pADPr. [source] Human Mus81 and FANCB independently contribute to repair of DNA damage during replicationGENES TO CELLS, Issue 10 2007Yuji Nomura Recent studies suggest a crucial role for homologous recombination (HR) in repairing replication-associated DNA lesions. In mammals, the Mus81 endonuclease and the Fanconi anemia (FA) pathway have been implicated in HR repair; however, their functional relationship has remained unexplored. Here, we knockout the genes for Mus81 and FANCB, a component of the FA core complex, in the human Nalm-6 cell line. We show that Mus81 plays an important role in cell proliferation to suppress cell death when FANCB is missing, indicating a functional linkage between Mus81 and the FA pathway. In DNA cross-link repair, roles for Mus81 and the FA pathway appear to have an overlapping function. Intriguingly, Mus81 and FANCB act independently in surviving exposure to camptothecin (CPT). Although CPT-induced FANCD2 and Mus81 foci co-localize with Rad51, loss of Mus81, but not FANCB, results in significantly decreased levels of spontaneous and CPT-induced sister chromatid exchanges (SCEs). In addition, Mus81, unlike FANCB, has no significant role in gene targeting as well as in repairing hydroxyurea (HU)-induced stalls of replication forks. Collectively, our results provide the first evidence for differential functions of Mus81 and the FA pathway in repair of DNA damage during replication in human cells. [source] Cytoplasmic splicing of tRNA in Saccharomyces cerevisiaeGENES TO CELLS, Issue 3 2007Tohru Yoshihisa The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae. These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre-tRNAs are bona fide intermediates for tRNA maturation in vivo. We isolated a new reversible allele of temperature-sensitive (ts) sen2 (HA-sen2-42), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA-sen2-42 cells accumulated large amounts of pre-tRNAs in the cytoplasm at a restrictive temperature, but the pre-tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse-chase/hybrid-precipitation techniques, we showed that the pre-tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae, pre-tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm. [source] Host factor Ebp1: Selective inhibitor of influenza virus transcriptaseGENES TO CELLS, Issue 2 2007Ayae Honda Influenza virus RNA polymerase is composed of three virus-coded proteins, and is involved in both transcription and replication of the negative-strand genome RNA. Subunit PB1 plays key roles in both the RNA polymerase assembly and the catalytic function of RNA polymerization. Using yeast two-hybrid screening, a HeLa cell protein with the molecular mass of 45 kDa was identified. After cloning and sequencing, this protein was identified to be Ebp1, ErbB3-binding protein. Epb1 specifically interacts with PB1 both in vitro and in vivo, and Epb1 contact site on PB1 was mapped at its binding site of transcription primers. Ebp1 was found to interfere with in vitro RNA synthesis by influenza virus RNA polymerase (3P complex), but no inhibition was observed for capped RNA endonuclease and RNA-cap binding, the intrinsic activities of RNA polymerase. Since inhibition was not observed against other nucleic acid polymerases tested, we propose that Ebp1 is a selective inhibitor of influenza viral RNA polymerase. Accordingly over-expression of Ebp1 interfered with virus production. The PB1-contact site on Ebp1 overlaps with the interaction site with ErbB3 (epidermal receptor tyrosine kinase), androgen receptor (AR) and retinoblastoma gene product (Rb), which are involved in controlling cell proliferation and differentiation. [source] VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like mannerGENES TO CELLS, Issue 7 2003Tomoyuki Fukuda Background: Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. Results: We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5, clb6, mutation reduces VDE-mediated DSBs. Conclusion: The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism. [source] A new HpaII PCR-RFLP within the porcine prolactin receptor (PRLR) gene and study of its effect on litter size and number of teatsJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2002L. PUTNOVÁ DNA polymorphism of the porcine prolactin receptor gene (PRLR) was investigated and used to study its effect on litter size and number of teats in pigs. By means of PRLR gene sequence homology in pig, human and other species, primers were designed for PCR amplification within 5, unknown (to date) part of the prolactin receptor gene in pigs. In this part of the gene, a new polymorphism with HpaII restriction endonuclease was detected. AluI polymorphism described before and our new HpaII polymorphism were used to study the associations with reproduction traits. The PCR restriction fragment length polymorphism (PCR-RFLP) method was used to genotype AluI and HpaII loci of the PRLR gene in line A with 83 sows of Landrace breed and in two lines (B and C) with 75 and 86 Large White sows, respectively. Statistical analysis of 1020 litters showed that AluI locus was associated with litter size mainly in Landrace and affected the first parities, while HpaII locus of the gene was associated with the same traits in Landrace and Large White pigs and mainly affected numbers of weaned of pigs. The magnitude of the effect varied by population with the effects exceeding two pigs per litter in Landrace line and 1 pig per litter in Large White populations. Ein neuer HpaII PCR-RFLP innerhalb des porcinen Prolaktionrezeptorgens (PRLR), und Zusammenhänge zur Wurfgröße und Zitzenzahl DNA-Polymorphismen im porcinen Prolaktionrezeptorgen (PRLR) wurden untersucht und für die Analyse von Einflüssen auf Wurfgröße und Zitzenzahl bei Schweinen verwendet. Auf der Basis der PRLR -Gensequenzhomologie zwischen Schwein, Mensch und anderen Spezies wurden Primer für die PCR-Amplifikation aus dem 5, Bereich des Prolaktionrezeptorgens abgeleitet, der bisher beim Schwein noch unbekannt ist. In diesem Teil des Gens wurde mittels HpaII-Restriktionsendonuklease ein neuer Polymorphismus dargestellt. AluI Polymorphismus und der neue HpaII Polymorphismus wurden für Assoziationsstudien in Bezug auf Reproduktionsmerkmale verwendet. Mittels PCR-RFLP wurden in Linie A 83 Sauen der Landrasse und die Linien B und C mit 75 bzw. 86 Large White Sauen unter Verwendung von AluI und HpaII am PRLR -Gen genotypisiert. Die statistische Analyse von 1.020 Würfen zeigte, dass der AluI-Polymorphismus insbesondere in der Landrasse mit der Wurfgröße assoziiert ist, sowie die ersten Trächtigkeiten beeinflusst, während der HpaII Polymorphismus die gleichen Merkmale in der Landrasse und Large White Schweinen und insbesondere die Zahl an abgesetzten Ferkeln beeinflusste. Die Auswirkungen des Effekts variierten innerhalb Population, wobei der Effekt 2 Ferkel je Wurf in der Landrasse-Linie und 1 Ferkel je Wurf in der Large White Populationen überstieg. [source] Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2000G. Frech A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates. [source] Apurinic/apyrimidinic endonuclease 1, p53, and thioredoxin are linked in control of aging in C. elegansAGING CELL, Issue 3 2010Andreas Schlotterer Summary Deletions in mitochondrial DNA (mtDNA) accumulate during aging. Expression of the Caenorhabditis elegans apurinic/apyrimidinic endonuclease 1 (APE1) ortholog exo-3, involved in DNA repair, is reduced by 45% (P < 0.05) during aging of C. elegans. Suppression of exo-3 by treatment with RNAi resulted in a threefold increase in mtDNA deletions (P < 0.05), twofold enhanced generation of reactive oxygen species (ROS) (P < 0.01), distortion of the structural integrity of the nervous system, reduction of head motility by 43% (P < 0.01) and whole animal motility by 38% (P < 0.05). Suppression of exo-3 significantly reduced life span: mean life span decreased from 18.5 ± 0.4 to 15.4 ± 0.1 days (P < 0.001) and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.1 days (P = 0.001). Additional treatment of exo-3 -suppressed animals with a mitochondrial uncoupler decreased ROS levels, reduced neuronal damage, and increased motility and life span. Additional suppression of the C. elegans p53 ortholog cep-1 in exo-3 RNAi-treated animals similarly decreased ROS levels, preserved neuronal integrity, and increased motility and life span. In wild-type animals, suppression of cep-1, involved in downregulation of exo-3, increased expression of exo-3 without a significant effect on ROS levels, preserved neuronal integrity, and increased motility and life span. Suppression of the C. elegans thioredoxin orthologs trx-1 and trx-2, involved in the redox chaperone activity of exo-3, overrides the protective effect of cep-1 RNAi treatment on neuronal integrity, neuronal function, mean and maximum life span. These results show that APE1/EXO-3, p53/CEP-1, and thioredoxin affect each other and that these interactions determine aging as well as neuronal structure and function. [source] Overexpression of human copper/zinc-superoxide dismutase in transgenic animals attenuates the reduction of apurinic/apyrimidinic endonuclease expression in neurons after in vitro ischemia and after transient global cerebral ischemiaJOURNAL OF NEUROCHEMISTRY, Issue 2 2005Purnima Narasimhan Abstract Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen,glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc-superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia. [source] Posttreatment with the Ca2+ -Mg2+ -dependent endonuclease inhibitor aurintricarboxylic acid abolishes genotoxic agent-induced nuclear condensation and DNA fragmentation and decreases death of astrocytes,JOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2008Huafei Lu Abstract DNA fragmentation and nuclear condensation are important nuclear changes in apoptosis. In this study we determined whether DNA fragmentation and nuclear condensation occur in astrocytes treated with 100,200 ,M of the genotoxic agent M -nitroso- N -nitroguanidine (MNNG). Our study also investigated the roles of Ca2+ -Mg2+ -dependent endonuclease (CME) in the MNNG-induced nuclear changes. We found that MNNG induced profound ATP depletion as well as marked nuclear condensation and DNA fragmentation in the cells. Both the nuclear condensation and the DNA fragmentation were abolished by posttreatment of the cells with the CME inhibitor aurintricarboxylic acid (ATA). The ATA posttreatment also significantly, but only partially, decreased MNNG-induced cell death. In contrast, pretreatment plus cotreatment with ATA did not affect either MNNG-induced nuclear condensation or cell death. Our study further suggests that ATA does not decrease the cytotoxicity of MNNG by directly inhibiting poly(ADP-ribose) polymerases. Collectively, our observations suggest that MNNG can induce both DNA fragmentation and nuclear condensation in astrocytes by a CME-dependent mechanism, which partially contributes to the genotoxic agent-induced cell death. Published 2008 Wiley-Liss, Inc. [source] Physiological and Biochemical Characteristics of Iranian Strains of Xanthomonas axonopodis pv. citri, the Causal Agent of Citrus bacterial Canker DiseaseJOURNAL OF PHYTOPATHOLOGY, Issue 2 2001M. Mohammadi Twenty-four strains of Xanthomonas axonopodis pv. citri (Xac), the causal agent of bacterial canker of citrus, isolated from Mexican lime (Citrus aurantifolia) and lemon (Citrus limon) in southern Iran, were characterized phenotypically. Strains were all pathogenic on C. aurantifolia. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed slight differences in soluble protein profiles among the strains. Based on host range specificity and phenotypic characteristics, representative strains were differentiated into two groups of Asiatic (A) and atypical Asiatic (aA) forms. DNA fingerprinting analysis using EcoRI as the restriction endonuclease showed a negligible difference in restriction pattern between the two groups. On the basis of isozymic analysis, the two groups were distinct with respect to superoxide dismutase (SOD) and esterase (EST) banding patterns. Plasmid DNA profile analysis showed that the bacterial strains were different from each other in terms of plasmid number and molecular weight. Phage typing study revealed that most of group A strains were susceptible to Cp1 and/or Cp2 and some were resistant to both phage types including the strain in aA group. Bacteriocin production test indicated that there was a variation among Xac strains using different indicators for each bacteriocin producer. It is concluded that the Iranian strains of Xac are heterogeneous and constitute a subgroup(s) within the pathotype A. Physiologische und biochemische Merkmale iranischer Stämme von Xanthomonas axonopodis pv. citri, dem Erreger des bakteriellen Zitruskrebses Vierundzwanzig Stämme von Xanthomonas axonopodis pv. citri, dem Erreger des bakteriellen Zitruskrebses, wurden von mexikanischen Sauren Limetten (Citrus aurantifolia) und Zitronen (Citrus limon) im Südiran isoliert und phänotypisch charakterisiert. Alle Stämme waren für C. aurantifolia pathogen. Eine SDS-PAGE-Analyse zeigte, daß zwischen den Stämmen geringfügige Unterschiede bei den Profilen der löslichen Proteine bestanden. Auf Grundlage der Spezifität des Wirtsspektrums und phänotypischer Merkmale wurden repräsentative Stämme in die zwei Gruppen asiatische (A) und atypische asiatische (aA) Formen eingeteilt. Eine Analyse mit DNA-Fingerabdrücken, wobei EcoRI als Restriktionsendonuclease diente, zeigte einen vernachlässigbar kleinen Unterschied bei den Restriktionsmustern der beiden Gruppen. Die Isoenzymanalyse ergab Unterschiede zwischen beiden Gruppen bezüglich der Bandenmuster von Superoxiddismutase (SOD) und Esterase (EST). Eine Analyse der Plasmid-DNA-Profile zeigte, daß die Bakterienstämme unterschiedliche Plasmidzahlen und verschiedene Molekülmassen aufwiesen. Eine Phagentypisierung ergab, daß die meisten Stämme der Gruppe A anfällig für Cp1 und/oder Cp2 waren; einige waren resistent gegen beide Phagentypen, darunter der Stamm in der aA-Gruppe. Ein Test der Bacteriocinproduktion ergab, daß die Xac -Stämme variierten; hier wurden verschiedene Indikatoren für jeden Bakteriocinbildner verwendet. Es wird gefolgert, daß die iranischen Stämme von Xac heterogen sind und eine oder mehrere Untergruppen innerhalb des Pathotyps A bilden. [source] Surgery-related shedding of breast cancer cells as determined by RT-PCR assayJOURNAL OF SURGICAL ONCOLOGY, Issue 4 2003Xi-Chun Hu MD Abstract Background and Objectives Surgery could result in the shedding of cancer cells into the circulation. These cells were investigated with reverse transcriptase-polymerase chain reaction (RT-PCR) assay for cytokeratin 19 (CK19) and ,-subunit of human chorionic gonadotropin (,-hCG). Patients and Methods Peripheral blood was sampled from 49 patients with breast cancer before operation (d,1), 1 day after operation (d1), and 7 days after operation (d7). Total RNA was extracted from peripheral blood mononuclear cells, followed by RT-PCR assay. The products for ,-hCG were digested with Sty I endonuclease. The patients were followed up for a median of 33 months for signs of recurrence and metastasis. Results The results for CK19 at d,1, d1, and d7 were 8.2, 20.4, and 10.2%, respectively. For ,-hCG, the corresponding results were 12.2, 26.5, and 16.3%, respectively. There was a higher positive rate in d1 samples than in d,1 samples for CK19 and ,-hCG (P,<,0.05 and P,=,0.092, respectively). Conversions of signals from being negative to positive were found in all stages. These did not demonstrate a statistical correlation with prognostic factors associated with a poor prognosis. Only two of the five recurrence occurred in the 15 patients with the signal conversions, while the other three occurred in the patients showing no signals in all samples. Conclusions Cancerous breast cells that enter into the blood circulation as a result of an operation are unlikely to be involved in the formation of metastatic deposits. J. Surg. Oncol. 2003;82:228,232. © 2003 Wiley-Liss, Inc. [source] Lead promotes abasic site accumulation and co-mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1,MOLECULAR CARCINOGENESIS, Issue 2 2007Daniel R. McNeill Abstract Lead is a widespread environmental toxin, found in contaminated water sources, household paints, and certain occupational settings. Classified as a probable carcinogen by the International Agency for Research on Cancer (IARC), lead promotes mutagenesis when combined with alkylating and oxidizing DNA-damaging agents. We previously reported that lead inhibits the in vitro repair activity of Ape1, the major endonuclease for repairing mutagenic and cytotoxic abasic sites in DNA. We investigated here whether lead targets Ape1 in cultured mammalian cells. We report a concentration-dependent inhibition of apurinic/apyrimidinic (AP) site incision activity of Chinese hamster ovary (CHO) AA8 whole cell extracts by lead. In addition, lead exposure results in a concentration-dependent accumulation of AP sites in the genomic DNA of AA8 cells. An increase in the oxidative base lesion 8-oxoguanine was observed only at high lead levels (500 µM), suggesting that non-specific oxidation plays little role in the production of lead-related AP lesions at physiological metal concentrations,a conclusion corroborated by "thiobarbituric acid reactive substances" assays. Notably, Ape1 overexpression in AA8 (hApe1-3 cell line) abrogated the lead-dependent increase in AP site steady-state levels. Moreover, lead functioned cooperatively to promote a further increase in abasic sites with agents known to generate AP sites in DNA (i.e., methyl methansulfonate (MMS) and hydrogen peroxide (H2O2)), but not the DNA crosslinking agent mitomycin C. Hypoxanthine guanine phosphoribosyltransferase (hprt) mutation analysis revealed that, whereas lead alone had no effect on mutation frequencies, mutagenesis increased in MMS treated, and to a greater extent lead/MMS treated, AA8 cells. With the hApe1-3 cell line, the number of mutant colonies in all treatment groups was found to be equal to that of the background level, indicating that Ape1 overexpression reverses MMS- and lead-associated hprt mutagenesis. Our studies in total indicate that Ape1 is a member of an emerging group of DNA surveillance proteins that are inhibited by environmental heavy metals, and suggest an underlying mechanism by which lead promotes co-carcinogenesis. Published 2006 Wiley-Liss, Inc., [source] Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assaysPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 1 2009Séverine Fontaine Abstract BACKGROUND: Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. RESULTS: The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations. Copyright © 2008 Society of Chemical Industry [source] Bound Transcription Factor Suppresses Photoproduct Formation in the NF-,B Promoter,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2001Rita Ghosh ABSTRACT The relationship between purified transcription factor p50 binding and ultraviolet light,induced DNA damage formation in the NF-,B promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam,Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters. [source] Heritable targeted mutagenesis in maize using a designed endonucleaseTHE PLANT JOURNAL, Issue 1 2010Huirong Gao Summary The liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I- CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I- CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium -mediated transformation of immature embryos, and transgenic T0 plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T0 plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant. [source] Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1THE PLANT JOURNAL, Issue 6 2000Zongrang Liu Summary To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium -mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3, splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues. [source] Structure of the restriction,modification controller protein C.Esp1396IACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2009N. Ball The controller protein of the Esp1396I restriction,modification (R,M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endonuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported; however, the structure of the free protein has not yet been determined. Here, the crystal structure of the free protein is reported, with seven dimers in the asymmetric unit. Two of the 14 monomers show an alternative conformation to the major conformer in which the side chains of residues 43,46 in the loop region flanking the DNA-recognition helix are displaced by up to 10,Å. It is proposed that the adoption of these two conformational states may play a role in DNA-sequence promiscuity. The two alternative conformations are also found in the R35A mutant structure, which is otherwise identical to the native protein. Comparison of the free and bound protein structures shows a 1.4,Å displacement of the recognition helices when the dimer is bound to its DNA target. [source] The structure of Vibrio cholerae extracellular endonuclease I reveals the presence of a buried chloride ionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2006Bjørn Altermark The crystal structure of a periplasmic/extracellular endonuclease from Vibrio cholerae has been solved at low and at neutral pH. Crystals grown at pH 4.6 and 6.9 diffracted to 1.6,Å (on BM01A at the ESRF) and 1.95,Å (on a rotating-anode generator), respectively. The structures of the endonuclease were compared with the structure of a homologous enzyme in V. vulnificus. The structures of the V. cholerae enzyme at different pH values are essentially identical to each other and to the V. vulnificus enzyme. However, interesting features were observed in the solvent structures. Both V. cholerae structures reveal the presence of a chloride ion completely buried within the core of the protein, with the nearest solvent molecule approximately 7,Å away. Magnesium, which is essential for catalysis, is present in the structure at neutral pH, but is absent at low pH, and may partly explain the inactivity of the enzyme at lower pH. [source] Crystallization and preliminary X-ray diffraction analysis of homing endonuclease I- Tsp061IACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Takahito Imagawa Two crystal forms, rhombohedral and hexagonal, of a homing endonuclease from Thermoproteus sp. IC-061 (I- Tsp0611) were obtained by the hanging-drop and sitting-drop method, respectively. The hexagonal crystals belong to space group P6322, with unit-cell parameters a = b = 111.4, c = 97.6,Å, and diffract to 3.2,Å resolution on beamline BL44 at SPring-8 (Harima, Japan). The rhombohedral crystals belong to space group R32, with unit-cell parameters a = b = 95.4, c = 192.9,Å, and diffract to 2.7,Å resolution using a Cu,K, rotating-anode generator with an R-AXIS VII detector. The crystal asymmetric unit contained one protein molecule and the solvent contents of the two crystal forms were estimated to be 68.3 and 67.6% by volume, respectively. [source] Crystallization and preliminary X-ray analysis of human endonuclease 1 (APE1) in complex with an oligonucleotide containing a 5,6-dihydrouracil (DHU) or an ,-anomeric 2,-deoxyadenosine (,dA) modified baseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Pascal Retailleau The multifunctional human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key enzyme involved in both the base-excision repair (BER) and nucleotide-incision repair (NIR) pathways. In the NIR pathway, APE1 incises DNA 5, to a number of oxidatively damaged bases. APE1 was crystallized in the presence of a 15-mer DNA containing an oxidatively damaged base in a single central 5,6-dihydrouracil (DHU)·T or ,-anomeric 2,-deoxyadenosine (,dA)·T base pair. Diffraction data sets were collected to 2.2 and 2.7,Å resolution from DNA-DHU,APE1 and DNA-,dA,APE1 crystals, respectively. The crystals were isomorphous and contained one enzyme molecule in the asymmetric unit. Molecular replacement was performed and the initial electron-density maps revealed that in both complexes APE1 had crystallized with a degradation DNA product reduced to a 6-mer, suggesting that NIR and exonuclease reactions occurred prior to crystallization. [source] Problem-solving test: Telomere replicationBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010József Szeberényi Terms to be familiar with before you start to solve the test: DNA replication, template, primer, linear DNA, antiparallel orientation, telomere, origin of replication, chromatid, replicon, short tandem repeats, Okazaki fragments, leading strand, lagging strand, ribozyme, promoter, enhancer, terminal transferase, DNA polymerases, reverse transcriptase, RNA polymerase, topoisomerase, retroviral vector, Southern blotting, restriction endonuclease. [source] Crystallization and preliminary X-ray analysis of flap endonuclease 1 (FEN1) from Desulfurococcus amylolyticusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Tomoko Mase Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes 5,-overhanging flaps in DNA repair and removes the RNA/DNA primer during maturation of the Okazaki fragment in lagging-strand DNA replication. FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with monoammonium dihydrogen phosphate as the precipitant at pH 8.3. X-ray diffraction data were collected to 2.00,Å resolution. The space group of the crystal was determined as the primitive hexagonal space group P321, with unit-cell parameters a = b = 103.76, c = 84.58,Å. The crystal contained one molecule in the asymmetric unit. [source] Purification, crystallization and data collection of the apoptotic nuclease endonuclease GACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Sei Mee Yoon Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving chromosomal DNA. EndoG is the main apoptotic endonuclease in the caspase-independent pathway. Mouse EndoG without the mitochondrial localization signal (amino-acid residues 1,43) was successfully overexpressed, purified and crystallized using a microbatch method under oil. The initial crystal (type I) was grown in the presence of the detergent CTAB and diffracted to 2.8,Å resolution, with unit-cell parameters a = 72.20, b = 81.88, c = 88.66,Å, , = 97.59° in a monoclinic space group. The crystal contained two monomers in the asymmetric unit, with a predicted solvent content of 46.6%. Subsequent mutation of Cys110 improved the stability of the protein significantly and produced further crystals of types II, III and IV with space groups C2, P41212 (or P43212) and P212121, respectively, in various conditions. This suggests the critical involvement of this conserved cysteine residue in the crystallization process. [source] Purification, crystallization and preliminary X-ray analysis of the HsdR subunit of the EcoR124I endonuclease from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007Mikalai Lapkouski EcoR124I is a multicomplex enzyme belonging to the type I restriction-modification system from Escherichia coli. Although EcoR124I has been extensively characterized biochemically, there is no direct structural information available about particular subunits. HsdR is a motor subunit that is responsible for ATP hydrolysis, DNA translocation and cleavage of the DNA substrate recognized by the complex. Recombinant HsdR subunit was crystallized using the sitting-drop vapour-diffusion method. Crystals belong to the primitive monoclinic space group, with unit-cell parameters a = 85.75, b = 124.71, c = 128.37,Å, , = 108.14°. Native data were collected to 2.6,Å resolution at the X12 beamline of EMBL Hamburg. [source] Clinical relevance of the homologous recombination machinery in cancer therapyCANCER SCIENCE, Issue 2 2008Kiyoshi Miyagawa Cancer chemotherapy and radiotherapy kill cancer cells by inducing DNA damage, unless the lesions are repaired by intrinsic repair pathways. DNA double-strand breaks (DSB) are the most deleterious type of damage caused by cancer therapy. Homologous recombination (HR) is one of the major repair pathways for DSB and is thus a potential target of cancer therapy. Cells with a defect in HR have been shown to be sensitive to a variety of DNA-damaging agents, particularly interstrand crosslink (ICL)-inducing agents such as mitomycin C and cisplatin. These findings have recently been applied to clinical studies of cancer therapy. ERCC1, a structure-specific endonuclease involved in nucleotide excision repair (NER) and HR, confers resistance to cisplatin. Patients with ERCC1-negative non-small-cell lung cancer were shown to benefit from adjuvant cisplatin-based chemotherapy. Imatinib, an inhibitor of the c-Abl kinase, has been investigated as a sensitizer in DNA-damaging therapy, because c-Abl activates Rad51, which plays a key role in HR. Furthermore, proteins involved in HR have been shown to repair DNA damage induced by a variety of other chemotherapeutic agents, including camptothecin and gemcitabine. These findings highlight the importance of HR machinery in cancer therapy. (Cancer Sci 2008; 99: 187,194) [source] Identification of a novel deletion in the OA1 gene: report of the first Spanish family with X-linked ocular albinismCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 5 2010Monica Martinez-Garcia PhD Abstract Background:, This study was undertaken to analyse the OA1 gene (GPR143) and its involvement in a Spanish family presenting with nystagmus, a common symptom of X-linked ocular albinism (XLOA). Methods:, DNA samples from the index case and eight relatives were analysed by multiplex ligation-dependent probe amplification (MLPA). Sequence analysis and restriction assay were used to confirm the results. In addition, an analysis of a STR located in intron 1 of the OA1 gene (OA-CA) was performed. Results:, The father of the proband presented with nystagmus, a feature consistent with XLOA. Mutation screening by multiplex ligation-dependent probe amplification and sequence analysis of the exon 2 of the OA1 gene led to the identification of the novel p.Glu129fsX35 (g.5815delA) mutation in two affected males and four carrier females. Three relatives were found to be non-mutated. The deletion detected resulted in a truncated protein 35 codons downstream and generated a new restriction site for the XcmI endonuclease. Additionally, microsatellite analysis showed co-segregation with the disease in the family. Conclusions:, A novel deletion in the OA1 gene was identified in a Spanish family with ocular albinism. The mutation detected is likely a loss-of-function alteration. To the best of our knowledge, we describe the first Spanish family known to present with XLOA due to mutations in the OA1 gene. [source] | ||||||||||||||||||||||||||||