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)

Our objective was to identify the endonuclease responsible for DNA degradation in the ovary and determine its localization relative to the developmental state of ovarian follicles. Immature rats were treated with diethylstilbestrol (DES; DES group), DES + eCG (eCG group) or DES + eCG + hCG (hCG group). Nuclei of the eCG and hCG but not the DES group contained a 32/34-kDA DNase I-like endonuclease activity that was Ca2+/Mg(2+)-dependent, stimulated by Mn2+, optimal at pH 7, and identified by anti-DNase I antibody. G-actin, Zn2+, dithiothreitol, aurintricarboxylic acid, and sodium aurothiomalate, but not iodoacetic acid, inhibited the activity. Addition of eCG nuclear protein extracts to nuclei from the DES group induced oligonucleosomal DNA fragmentation, which could be prevented by pretreatment of the extracts with anti-DNase I antibody. DNase I was immunolocalized in nuclei of healthy luteal cells, antral follicle granulosa cells, oocytes of preantral follicles, atretic preantral follicles, and testicular spermatogonia, but was not observed in granulosa cells of preantral follicles, theca cells, antral follicle oocytes, or testicular spermatocytes. Nuclear extracts of rat kidney, liver, and spleen, and bovine, chicken, and human ovaries displayed DNase I-like activity. These results suggest that an endonuclease indistinguishable from DNase I is responsible for ovarian apoptotic DNA degradation.
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PMID:Identification and localization of deoxyribonuclease I in the rat ovary. 931 85

Complementation group G of xeroderma pigmentosum (XPG) is one of the most rare and pathophysiologically heterogeneous forms of this inherited disease. XPG patients exhibit varying phenotypes, from having a very mild defect in DNA repair to being severely affected, and a few cases are also associated with the neurological degeneracy and growth retardation of Cockayne's syndrome. The XPG gene encodes a 134-kDa nuclear protein that is essential for the incision steps of nucleotide excision repair. XPG protein contains a putative helix-loop-helix (HLH) motif in the region that is most conserved among the members of structure-specific endonuclease family. To establish the functional significance of the HLH motif, we used several approaches, including theoretical modeling, functional complementation assay, structure-specific endonuclease assay, and DNA binding assay. A secondary structure of the motif was predicted by energy minimization and the Monte Carlo simulation and empirically proven using the circular dichroism to contain a high content of alpha-helix. When an XPG mutant lacking the HLH was overexpressed in UV135 cells, which have defects in the hamster homolog of XPG, the mutant gene failed to confer to the hamster cells the resistance to UV light. A recombinant XPG protein lacking the HLH motif was purified from insect cells and tested for a structure-specific endonuclease activity. The mutant protein failed to cleave the flap strand. A recombinant peptide containing the HLH (amino acids 758-871) was expressed in and purified from bacteria, tested for DNA binding activity, and found to bind to a DNA substrate with the flap structure. These results suggest that the HLH motif is required for the catalytic and DNA binding activities of XPG.
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PMID:Characterization of a putative helix-loop-helix motif in nucleotide excision repair endonuclease, XPG. 934 28

A cell-free plasmid reactivation assay was used to determine the fidelity of DNA double-strand break (DSB) repair in a panel of eight DSB repair-proficient human tumour cell lines. Nuclear protein extracts derived from radiosensitive tumour cells were less capable of correctly rejoining EcoRI-induced DSBs than were similar extracts from radioresistant tumour cells. Linear regression analysis suggests that there was a significant (r2 = 0.84, P = 0.001, d.f. = 6) correlation between the fidelity of DSB rejoining and the SF2 values of the cell lines studied. This cell-free assay is clearly sensitive to differences in the nuclear protein composition that reflect the clinically relevant radiosensitivity of these cell lines. The fact that our cell-free assay yielded similar results to previous studies that used intracellular plasmid reactivation assays suggests that those differences in DSB mis-rejoining frequencies in radiosensitive and radioresistant cell lines may be due to inherent differences in nuclear protein composition and are not directly attributable to differences in proliferation rates between cell lines. The underlying cause for this association between DSB mis-rejoining frequencies and radiosensitivity is presently unknown, however restriction endonuclease mapping and polymerase chain reaction (PCR) amplification analysis revealed that approximately 40% of the mis-rejoined DSBs arose as a result of the deletion of between 40 and 440 base pairs. These data raise the possibility that the radiosensitivity of DSB repair-proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways.
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PMID:Differential level of DSB repair fidelity effected by nuclear protein extracts derived from radiosensitive and radioresistant human tumour cells. 940 Sep 40

A rat gene, designated DNaseY, encoding a 36 kDa endonuclease was identified and cloned. Sequence analysis of the cDNA showed it to be the rat homologue of human DNAS1L3. The DNaseY gene product had 42% identity to DNaseI, including conserved critical active site residues, the essential disulfide bridge, the calcium binding domain, and a signal peptide, as well as 2 of the 3 signature boxes. Significantly, DNaseY had 2 nuclear localization signals and was more basic (pI 9.5) than DNaseI (pI 4.8). The DNaseY gene contained a number of exons similar to that of DNaseI, separated by much larger introns, resulting in a gene of >17 kb compared to <4 kb gene of DNaseI. The 36 kDa DNaseY gene product was catalytically inactive but was converted to an active 33 kDa endonuclease following processing of the hydrophobic signal peptide. Antibody generated against peptides representing the predicted amino acid sequence of DNaseY cross-reacted with a 33 kDa nuclear protein which possessed endonucleolytic activity. The enzyme was active over a broad pH range (optimum pH 7-8), was Ca2+/Mg2+-dependent, was inhibited by Zn2+, and was capable of both single- and double-stranded DNA cleavage, producing DNA fragments with 3'-OH ends. Furthermore, the DNaseY gene was expressed constitutively in all cells and tissues tested, but it was not transcriptionally up-regulated in apoptotic cells. All these features were consistent with a role in the early stages of apoptotic DNA fragmentation.
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PMID:DNaseY: a rat DNaseI-like gene coding for a constitutively expressed chromatin-bound endonuclease. 966 19

We have tested the specific hypothesis that the pathway of nuclear collapse in apoptosis is governed by the early attack on active chromatin at spatially restricted nuclear sites. Cell death in PC12 pheochromocytoma cells deprived of serum growth factors, in HL-60 leukemic cells treated with inhibitors of protein or RNA biosynthesis, and in U937 histiocytic lymphoma cells exposed to the cytokine tumor necrosis factor alpha showed a common mechanism in the targeting of DNA for degradation. An incorporation assay with labeled nucleotide revealed an early selective nicking in peripheral nuclear chromatin with concomitant diminution in the amount of immunoreactive lamin B protein. This was followed by a phase of more extensive cleavages, continued nuclear protein loss, chromatin collapse, and fragmentation of nuclei. The spatial restriction of early cleavages is similar to the nicking obtained by the application of exogenous DNase I to fixed nuclei of normal cells and to that obtained in the activation of the endogenous endonuclease of liver nuclei by Ca2+. These similarities suggest that, in apoptosis, activation of an endonuclease preferentially recognizing a specific chromatin configuration, such as that of active (DNase I-sensitive) genes, underlies the early spatial demarcation of cleavages.
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PMID:Preferential sites of early DNA cleavage in apoptosis and the pathway of nuclear damage. 1021 26

At first sight a protein that is enriched in extracts prepared from nuclei by means of biochemical methods can be considered to be a nuclear protein in vivo. Although this assumption will hold true for most of the analyzed proteins, it could also lead to false interpretations. We analyzed the subcellular distribution of endogenous and plasmid-borne proteins derived from the retrotransposon TRE5-A of Dictyostelium discoideum. In biochemical fractionation experiments the proteins encoded by TRE5-A open reading frame 1 (ORF1p) and the putative endonuclease encoded in ORF2 (ENp) were found in the detergent-insoluble material containing the nuclei. However, salt extraction of isolated nuclei did not considerably release the TRE5-A proteins. Instead, the TRE5-A proteins were strongly enriched in a fraction that contained the chromosomal DNA after removal of most cytoskeletal and histone proteins. These observations implied that ORF1p and ENp were both attached to chromatin in vivo, but this conclusion was disproved by the expression of genetic fusions of green fluorescent protein with either ORF1p or ENp. We show conclusive evidence that both fusion proteins were located as large aggregates of native protein in the cytoplasm of D. discoideum cells.
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PMID:On the problem of establishing the subcellular localization of Dictyostelium retrotransposon TRE5-A proteins by biochemical analysis of nuclear extracts. 1152 35

Triplex stabilization by poly(L-lysine)-graft-dextran copolymer within a mammalian gene promoter inhibits the DNA binding activity of nuclear proteins from HeLa cells as well as restriction endonuclease cleavage at physiological pH and ionic conditions in vitro. Electrophoretic mobility shift assays using a 30-mer homopurine-homopyrimidine stretch (located between -170 and -141 bp) of rat alpha 1 (I) collagen gene promoter reveal that the copolymer, at its wide range of charge ratio with DNA, stabilizes triplex DNA and enhances triplex-specific inhibition of the protein-DNA interaction. When the triplex-forming region (located between -165 and -146 bp) of the promoter is engineered at the Bam H1 and Pst 1 sites of a plasmid DNA, copolymer-mediated triplex stabilization also remarkably competes endonuclease activity of BamH1. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of spermine and benzo[e]pyridoindole. Our results indicate that the copolymer, regardless of the length of the target duplex, stabilizes triplexes for significant inhibition of protein-DNA interaction and endonuclease activity. Since stable triplex formation within a short region out of a long native duplex is a prerequisite to confer the therapeutic potential of antigene strategy, triplex stabilization on a long target duplex and inhibition of nuclear protein-DNA interaction may open the possible in vivo applicability of the copolymer.
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PMID:Inhibition of sequence-specific protein-DNA interaction and restriction endonuclease cleavage via triplex stabilization by poly(L-lysine)-graft-dextran copolymer. 1171 99

The major human AP-endonuclease 1 (APE1) is a multifunctional protein that plays a central role in the repair of damaged DNA by acting as a dual-function nuclease in the base excision repair pathway. This enzyme was also independently identified as a redox activator of AP-1 DNA-binding activity and has subsequently been shown to activate a variety of transcription factors via a redox mechanism. In a third distinct role, APE1 was identified as a component of a trans-acting complex that acts as a repressor by binding to the negative calcium responsive elements (nCaRE)-A and nCaRE-B, which were first discovered in the promoter of the human parathyroid gene and later in the APE1 promoter itself. Here we show that the nuclear protein complex which binds to the nCaRE-B2 of the hAPE1 gene contains APE1 itself and the heterogeneous nuclear ribonucleoprotein L (hnRNP-L). The interaction between the APE1 and hnRNP-L proteins does not require the presence of nCaRE-B2. Our results support the possibility that the APE1 gene is down-regulated by its own product, which would be the first such example of the regulation of a DNA repair enzyme, and identify a novel function of hnRNP-L in transcriptional regulation.
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PMID:Human AP-endonuclease 1 and hnRNP-L interact with a nCaRE-like repressor element in the AP-endonuclease 1 promoter. 1180 97

The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease-helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1(+), encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5' to 3' DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the completion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.
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PMID:The fission yeast pfh1(+) gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase. 1240 64

Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca2+,Mg2+-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mg2+) > Mn2+ = (Ca2+ + Mn2+) > (Mg2+ + EGTA) > Ca2+. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn(2+) inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca2+,Mn2+-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mn2+) > (Ca2+ + Mg2+) > Mn2+ > (Mg2+ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.
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PMID:Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius. 1288 41


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