Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A sequence of 1624 bp 5' to the apurinic/apyrimidinic (AP) endonuclease structural gene of Dictyostelium discoideum (APEA) has been inserted upstream of the luciferase reporter gene in pVTL2, an autonomously replicating nuclear plasmid in this organism. Cells transformed with this plasmid, designated pVTL-AL, displayed strong luciferase induction during treatment with the DNA-damaging agent bleomycin. For example, a luciferase activity of 45-fold above the constitutive level was observed for 20 hours of growth in axenic medium with 0.002 U/mL of bleomycin. The response was bleomycin concentration-dependent. Cell survival was greater than 90% for all treatments. The level of luciferase expression was highly dependent on the cell growth conditions, with the greatest induction observed for stationary phase axenically-grown cells. This effect may be related to a variation of plasmid copy number with growth conditions.
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PMID:Dictyostelium discoideum plasmid containing an AP-endonuclease upstream sequence: bleomycin induction of a luciferase reporter. 1174 61

Cancer development requires the accumulation of numerous genetic changes, which are believed to initiate through the presence of unrepaired lesions in the genome. In the absence of proficient repair, genotoxic agents can lead to crucial mutations of vital cellular genes via replication of damaged DNA. Many cell cycle regulatory proteins are known to modulate the repair capacity and consequently the fate of cells. We and others have recently shown that p53 tumor suppressor gene product is required for efficient global genomic repair (GGR) but not the transcription coupled repair (TCR) of the nucleotide excision repair (NER) sub-pathways. In order to discern the nature of the p53 modulation to be direct or indirect through a downstream mediator, we have investigated the processing of UV radiation induced lesions in human colon carcinoma, HCT116 cells expressing wild-type p53 but having different p21(waf1cip1) (hereafter p21) genotypes (p21+/+, p21+/-, p21-/-). Following 20 J/m(2) UV, all the three cell lines showed rapid increase in p53 protein but the accompanying increase in the expression of its downstream target protein p21 could only be seen in p21+/+ and p21+/- cells and not in p21-/- cells. Nevertheless, an absence of detectable p21 protein in deficient cells had no demonstrable effect on DNA repair response to UV irradiation, as measured by an immunoassay to detect removal of UV photoproducts from genomic DNA (GGR) and by individual strand specific removal of endonuclease-sensitive CPD from a target gene fragment (TCR). Introduction of cytomegalovirus (CMV)-driven luciferase reporter plasmid, UV damaged in vitro, into the un-irradiated cells of varying p21 background, revealed a relatively small but statistically significant decrease in the reporter expression in the host p21-/- as compared with p21+/+ and p21+/- HCT116 cells. Super-expression of p21 protein upon reintroduction of p21 expression construct, showed an enhanced recovery of UV damaged reporter activity that was not greatly different from a similar enhancement observed with undamaged plasmid reporter DNA. Taken together, the results indicate that (i) the p21 protein does not have a significant role in the repair of genomic DNA at chromosomal level; (ii) the well-established p53 dependent modulation of NER is distinct and independent of its cell cycle checkpoint function; and (iii) the reproducible enhancing effect of p21 expression observed through host cell reactivation (HCR) of extrachromosomal DNA is mainly attributable to an effect exerted on transcription rather than repair.
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PMID:Human cells deficient in p53 regulated p21(waf1/cip1) expression exhibit normal nucleotide excision repair of UV-induced DNA damage. 1189 54

HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5' to 3' exonuclease and flap structure-specific endonuclease activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter fragment extending from -6240 to +1600bp exhibits cell-type specific activity in transient transfection assays. This fragment directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified a fragment spanning -688 to +1600bp that exhibits full transcriptional activity while a slightly shorter fragment from -658 to +1600bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that interact with -687 to -681bp and -665 to -658bp elements. Oligonucleotide competition and antibody disruption studies determined that the transcription factor CREB-1 recognizes the -687 to -681bp element, while transcription factors Sp1 and Sp3 recognize the -665 to -658bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.
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PMID:Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity. 1253 89

Multiply damaged sites (MDS) are defined as greater than/equal to two lesions within 10-15 bp and are generated in DNA by ionizing radiation. In vitro repair of closely opposed base damages > or =2 bp apart results in a double strand break (DSB). This work extends the in vitro studies by utilizing clusters of uracil DNA damage as model lesions to determine whether MDS are converted to DSBs in bacteria. Lesions were positioned within the firefly luciferase coding region, transformed into bacteria (wild-type, uracil DNA glycosylase-deficient, ung-, or exonuclease III and endonuclease IV-deficient, xth-nfo-) and luciferase activity measured following repair. DSB formation was expected to decrease activity. Two closely opposed uracils separated by < or =7 bp decreased luciferase activity in wild-type and xth-nfo-, but not ung- bacteria. Growth of bacteria to obtain plasmid-containing colonies demonstrated that the plasmid was destroyed following the mis-repair of two uracils positioned 7 bp apart. This study indicates a DSB is formed when uracil DNA glycosylase initiates repair of two closely opposed uracils < or =7 bp apart, even in the absence of the major apurinic endonucleases. This work supports the in vitro studies and demonstrates that DNA repair is not always advantageous to cells.
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PMID:Repair of clustered uracil DNA damages in Escherichia coli. 1288 18

PMR1 is a polysome-associated mRNA endonuclease that initiates the destabilization of albumin mRNA. The current study examined whether endonuclease-mediated mRNA decay involved the selective binding of PMR1 to substrate mRNA on polysomes. PMR1 is uniformly distributed throughout the cytoplasm on polysomes and in lighter complexes and does not colocalize in cytoplasmic foci with Dcp1. Deletion mutagenesis identified polysome-targeting domains in the N and C termini of PMR1, either of which could target GFP to polysomes. Selectivity in targeting to polysome-bound substrate mRNP was determined by testing the ability of full-length PMR1 or protein lacking targeting domains to recover albumin and luciferase mRNA from dissociated polysomes. Only PMR1 bearing intact polysome-targeting domains selectively recovered albumin mRNA, and polysome targeting of both protein and substrate was required for the efficient degradation of albumin mRNA. Thus, endonuclease-mediated mRNA decay occurs on a polysome-bound complex containing PMR1 and its substrate mRNA.
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PMID:Endonuclease-mediated mRNA decay involves the selective targeting of PMR1 to polyribosome-bound substrate mRNA. 1514 93

Eukaryotic small heat shock proteins (shps) act as molecular chaperones by binding to denaturing proteins, preventing their heat-induced aggregation and maintaining their solubility until they can be refolded back to their normal state by other chaperones. In this study we report on the functional characterization of a developmentally regulated shsp, hsp30, from the American bullfrog, Rana catesbeiana. An expression vector containing the open reading frame of the hsp30 gene was expressed in Escherichia coli. Purified recombinant hsp30 was recovered as multimeric complexes and was composed of a mixture of alpha-helical and beta-sheet-like structures as determined by circular dichroism analysis. Hsp30 displayed chaperone activity since it inhibited heat-induced aggregation of citrate synthase. Furthermore hsp30 maintained heat-treated luciferase in a folding competent state. For example, heat denatured luciferase when microinjected into Xenopus oocytes did not regain enzyme activity whereas luciferase heat denatured with hsp30 regained 100% enzyme activity. Finally, hsp30 protected the DNA restriction endonuclease, PstI, from heat inactivation. PstI incubated alone at 42 degrees C lost its enzymatic function after 1 h whereas PstI supplemented with hsp30 accurately digested plasmid DNA after 4 h at the elevated temperature. These results clearly indicate a molecular chaperone role for R. catesbeiana hsp30.
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PMID:Molecular chaperone function of the Rana catesbeiana small heat shock protein, hsp30. 1552 66

Inducible promoter fusions are commonly employed to study the biological functions of genes as well as to investigate mechanisms of transcription regulation. A concern for many studies of heterologous gene expression is that steady state transcription may be too high under non-inducing conditions, producing undesired phenotypes prior to induction. Fusions containing the galactose-inducible GAL1 promoter joined to PvuII, a bacterial DNA endonuclease gene, are toxic to yeast cells even under non-inducing conditions, i.e., in glucose media. This toxicity was utilized in conjunction with PCR-based mutagenesis of the GAL1 regulatory region to isolate mutant promoters that retained high inducibility but exhibited reduced basal level expression. The Mig1 repressor binding and putative TATA box regions were unchanged among four mutant promoters examined in detail. However, each promoter contained one or more mutations within previously identified binding sites for the Gal4 activator protein. Genetic assays developed to monitor GAL1p::I-SceI endonuclease-induced recombination demonstrated that basal expression from two of the new promoters (designated GAL1-V4 and GAL1-V10) was strongly reduced. These experiments and additional quantitative luciferase reporter gene assays demonstrate the utility of the approach for identifying promoters that permit more tightly controlled gene expression.
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PMID:Use of a restriction endonuclease cytotoxicity assay to identify inducible GAL1 promoter variants with reduced basal activity. 1628 30

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.
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PMID:Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage. 1820 37

Clustered lesions are defined as >or=two lesions within 20 bps and are generated in DNA by ionizing radiation. In vitro studies and work in bacteria have shown that attempted repair of two closely opposed lesions can result in the formation of double strand breaks (DSBs). Since mammalian cells can repair DSBs by non-homologous end-joining (NHEJ), we hypothesized that NHEJ would repair DSBs formed during the removal of clustered tetrahydrofurans (furans). However, two opposing furans situated 2, 5 or 12 bps apart in a firefly luciferase reporter plasmid caused a decrease in luciferase activity in wild-type, Ku80 or DNA-PKcs-deficient cells, indicating the generation of DSBs. Loss of luciferase activity was maximal at 5 bps apart and studies using siRNA implicate the major AP endonuclease in the initial cleavage. Since NHEJ-deficient cells had equivalent luciferase activity to their isogenic wild-type cells, NHEJ was not involved in accurate repair of clustered lesions. However, quantitation and examination of re-isolated DNA showed that damage-containing plasmids were inaccurately repaired by Ku80-dependent, as well as Ku80-independent mechanisms. This work indicates that not even NHEJ can completely prevent the conversion of clustered lesions to potentially lethal DSBs, so demonstrating the biological relevance of ionizing radiation-induced clustered damage.
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PMID:DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining. 1865 25

Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a ubiquitous multifunctional protein that possesses both DNA-repair and redox regulatory activities. Although it was originally identified as a DNA-repair enzyme, accumulating evidence supports a role of APE1/Ref-1 in tumor development. To investigate association between APE1/Ref-1 polymorphisms and lung cancer risk in Chinese populations, we first genotyped three variants of APE1/Ref-1 and found a -141 T-to-G variant (rs1760944) in the promoter associated with decreased risk of lung cancer [odds ratio (OR) = 0.62 for GG; P=0.043]. Similar results were obtained in a follow-up replication study. Combined data from the two studies comprising a total of 1072 lung cancer patients and 1064 cancer-free control participants generated a more significant association (P=0.002). We observed lower APE1/Ref-1 mRNA levels in the presence of the protective G allele in human peripheral blood mononuclear cells and normal lung tissues. The -141G-allele-promoter construct exhibited decreased luciferase reporter gene expression. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that the -141G allele impaired the binding affinity of some transcription factor, accounting for lower APE1/Ref-1-promoter activity. Supershift assays further revealed that the protein of interest was octamer-binding transcription factor-1 (Oct-1). Chromatin immunoprecipitation reconfirmed binding of Oct-1 to the APE1/Ref-1 -141-promoter region. We also found that Oct-1 conferred attenuated transactivation capacity toward the -141G variant by exogenously introducing Oct-1. These data indicate that genetic variations in APE1/Ref-1 may modify susceptibility to lung cancer and provide new insights into an unexpected effect of APE1/Ref-1 on lung carcinogenesis.
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PMID:Functional characterization of a promoter polymorphism in APE1/Ref-1 that contributes to reduced lung cancer susceptibility. 1954 47


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