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)

The human leukemia cell line, HL60 is very sensitive to various apoptotic stimuli and p53-null. The death-related cysteine proteases of the caspases family play a central role in the execution phase of apoptosis, and we recently reported the importance of serine protease activation in camptothecin-induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CED-3-related cysteine proteases) and serine proteases in cell death induced by the topoisomerase I inhibitor, camptothecin, in HL60 cells and in a cell-free system. We found that CPP32 is activated during camptothecin-induced apoptosis, and that N-benzyloxycarbony-Val-Ala-Asp (O-methyl) -fluoromethyketone (Z-VAD-fmk), a cell permeable caspase inhibitor blocks all features of apoptosis: morphological changes, cleavage of caspase 3 (CPP32/Yama/Apopain) and poly(ADP-ribose) polymerase, lamin B degradation and DNA fragmentation. However, Z-VAD-fmk and two other ICE/CED-3 inhibitors, YVAD-CHO and DEVD-CHO, were inactive in a cell-free system reconstituted from nuclei of untreated HL60 cells and cytosol from camptothecin-treated cells, suggesting that caspases are not required for endonuclease activation or lamin B cleavage in the cell-free system. By contrast, the serine protease inhibitors, 3,4-dichloroisocoumarin (DCI) and L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone (TPCK), abolished the apoptosis-associated biochemical changes induced by camptothecin both in whole cells and in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32 and serine proteases are activated in camptothecin-induced apoptosis.
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PMID:Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases. 926 76

The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5'-CAGCTG-3') through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA- or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.
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PMID:Catalytic and DNA binding properties of PvuII restriction endonuclease mutants. 932 3

The 9B, 123, 788/8 Pseudomonas syringae phages were investigated. PAAG electrophoretic profiles of phage proteins were identical for all three phages except the minor polypeptide having molecular weight 35,000 Da. The band corresponding to this protein was present only in 9B and 788/8 phage protein profiles. Amino acid composition of phage proteins varied insignificantly showing prevalence of Asp, Glu, Ala, Leu. Phage DNA fragments electrophoresis, carried out after processing with specific endonuclease Hind III, made it possible to evaluate common restriction sites in phage genomes. Genome molecular weight was equal to 15 mda for 9B phage and to 14 mDa for 123 and 788/8 phages. The analysis of phage growth cycle showed that latent period consisted of 50 min at 20 degrees C and the yield equalled to 70 virions per infected bacterium cell. The similarity of the phages' features suggests their broad spreading in the environment.
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PMID:[The properties of the proteins and nucleic acids of 3 Pseudomonas syringae phages]. 948 14

The molecular mechanism of cell death induced by 5-Fluoro-2'-deoxyuridine (FUdR) was investigated. FUdR caused cell death to induce dNTP pool imbalance and following DNA double strand breaks in mouse mammary tumor FM3A cells. We isolated a new endonuclease from FUdR-treated cells, named endonuclease S, that played an important role in FUdR-induced cell death. Cells treated with FUdR showed intracellular acidification before cell death formation. We observed that the endonuclease S in acidic cells may lead the DNA fragmentation. On the other hand, we observed that protease inhibitors (such as TLCK, TPCK, PMSF, p-APMSF, Pefabloc SC and Z-Asp-CH2-DCB) blocked intracellular acidification, DNA fragmentation and FUdR-induced cell death. But the inhibitors did not affect dNTP pool imbalance in the cells. These results suggest that proteases act at the point of downstream of dNTP pool imbalance and upstream of the intracellular acidification.
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PMID:The molecular mechanisms of 5-fluoro-2'-deoxyuridine induced cell death. 958 36

An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).
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PMID:Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis. 963 81

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.
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PMID:Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. 969 73

Superposition of the PI-SceI and I-CreI homing endonuclease three-dimensional x-ray structures indicates general similarity between the I-CreI homodimer and the PI-SceI endonuclease domain. Saddle-shaped structures are present in each protein that are proposed to bind DNA. At the putative endonucleolytic active sites, the superposition reveals that two lysine (Lys-301 and Lys-403 in PI-SceI and Lys-98 and Lys-98' in I-CreI) and two aspartic acid residues (Asp-218 and Asp-326 in PI-SceI and Asp-20 and Asp-20' in I-CreI) are related by 2-fold symmetry. The critical role of Lys-301, Asp-218, and Asp-326 in the PI-SceI reaction pathway was reported previously. Here, we demonstrate the significance of the active-site symmetry by showing that alanine substitution at Lys-403 reduces cleavage activity by greater than 50-fold but has little effect on the DNA binding activity of the mutant enzyme. Substitution of Lys-403 with arginine, which maintains the positive charge, has only a modest effect on activity. Interestingly, even though the Lys-301 and Lys-403 residues display pseudosymmetry, PI-SceI mutant proteins with substitutions at these positions have different behaviors. The presence of similar basic and acidic residues in many LAGLIDADG homing endonucleases suggests that these enzymes use a common reaction mechanism to cleave double-stranded DNA.
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PMID:Identification of Lys-403 in the PI-SceI homing endonuclease as part of a symmetric catalytic center. 980 21

We have previously implicated deoxyribonuclease II (DNase II) as an endonuclease responsible for DNA digestion during apoptosis. The full-length human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 kDa larger than commercially supplied enzyme, which has been proteolytically cleaved at an internal aspartate residue. The gene is located at chromosome 19p13.2, and has no significant homology to other human proteins, but has >30% identity to three predicted genes in Caenorhabditis elegans. To determine whether overexpression of DNase II induces apoptosis in Chinese hamster ovary cells, the cDNA was cotransfected with a plasmid encoding green fluorescent protein. Within 24 h, a significant proportion of green fluorescent protein-positive cells contained condensed chromatin, whereas vector-only controls remained viable. Considering that DNase II is normally active only at low pH, it was surprising that transfection induced chromatin condensation. To confirm that transfection was not activating another endonuclease, cells were incubated with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone; this failed to inhibit chromatin condensation induced by DNase II. These results demonstrate that DNase II acts downstream of caspase activation and that it may be activated by an as yet unknown mechanism to induce DNA digestion during apoptosis.
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PMID:The cloning and expression of human deoxyribonuclease II. A possible role in apoptosis. 981 84

Endonucleolytic DNA fragmentation is the common end point and the prevailing indicator of apoptosis. We have identified a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. We purified NUC70 to homogeneity and generated a rabbit polyclonal antibody to distinguish it from previously identified nucleases. Biochemical characterization of isolated NUC70 demonstrates that it is Ca2+/Mg2+-dependent and active over a pH range of 6-8. When incubated with isolated HeLa nuclei, NUC70 was capable of generating internucleosomal DNA fragmentation. This endonucleolytic activity was inhibited by Zn2+, aurintricarboxylic acid, N-ethylmaleimide, spermine, and iodoacetamide. Western immunoblots using the anti-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indicate that NUC70 expression and activity is restricted to human hematopoietic cells. No such activity was detected in human epithelial cell lines or murine hematopoietic cells. We also observed no difference in levels of NUC70 expression between apoptotic and nonapoptotic cells, suggesting that activation of NUC70 may be by posttranslational modification. We demonstrate that NUC70 activity is diminished in cells pretreated with the caspase inhibitors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course studies of cytoplasmic and nuclear endonuclease activities during apoptosis show that NUC70 is a cytoplasmic endonuclease that is translocated to the nucleus after the initiation of apoptosis. We confirmed this with immunostaining studies using anti-NUC70 antibody. These results demonstrate that NUC70 is an endogenous cytoplasmic endonuclease that is activated during apoptosis in a caspase-dependent mechanism.
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PMID:Isolation and characterization of NUC70, a cytoplasmic, hematopoietic apoptotic endonuclease. 985 8

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA substrates that have the 5'-end of the nick expanded into a single-stranded tail, and it is involved in processing Okazaki fragments during DNA replication. Magnesium is a cofactor required for nuclease activity. We used small-angle x-ray scattering to obtain global structural information pertinent to nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1. 34-mer DNA flap complex, and the D181A.34-mer DNA flap complex. The D181A mutant, which has Asp-181 replaced by Ala, selectively binds to the flap structure, but has lost its cleaving activity. Asp-181 is thought to be involved in Mg2+ binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173-9176). Our data indicate that FEN-1 and the D181A mutant each have a radius of gyration of approximately 26 A, and the effect of Mg2+ on the scattering from the proteins alone is insignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5'-flap structure. The formation of the flap conformation of the DNA substrate was evident by both the extrapolated Io scattering and radius of gyration and was supported by NMR spectrum and nuclease assays. In the absence of magnesium, the FEN-1.34-mer DNA flap complex has an Rg value of approximately 34 A, whereas the D181A.34-mer DNA flap complex self-associates, suggesting that a significant protein conformational change occurs by addition of the flap DNA substrate and that Asp-181 is crucial for proper binding of the protein to the DNA substrate. A time course change in the scattering profiles arising from magnesium activation of the FEN-1.34-mer DNA flap complex is consistent with the protein completely releasing the DNA substrate after cleavage.
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PMID:Structural changes measured by X-ray scattering from human flap endonuclease-1 complexed with Mg2+ and flap DNA substrate. 988 Apr 91


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