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)

Agents that induce DNA strand breaks evoke a drop in the NAD content in mouse thymocytes. A decrease in the endogenous NAD content that occurs immediately after gamma-irradiation of thymocytes is entirely attributed to the activation of poly(ADP-ribosylation). The addition of 5 mM benzamide before irradiation prevents the postirradiation drop of the NAD level but has no effect on chromatin degradation and cell death. In contrast to liver nuclei, pre-incubation of mouse thymic nuclei with NAD had no effect on the subsequent chromatin endonucleolysis by Ca2+/Mg2+-dependent endonuclease. It is suggested that the NAD-poly(ADP-ribose) polymerase system is probably not the trigger in the radiation-induced programmed death of mouse thymocytes, but may merely be indicative of the radiation response of these cells.
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PMID:Is the NAD-poly (ADP-ribose) polymerase system the trigger in radiation-induced death of mouse thymocytes? 257 Aug 13

The previously reported extensive DNA strand breakage in resting murine splenic lymphocytes is not an artifact of the extraction or assay procedure. The benzamide inhibitors of poly(ADP ribose) synthetase (pADPRS), such as 5-methoxybenzamide (MBA), had been shown to block the strand break repair occurring within 2 h of activation of splenic lymphocytes by the mitogen concanavalin A (conA); the inhibitors also blocked early events in proliferation, such as blast formation, as well as entry into S phase. Inhibitors of pADPRS blocked lymphocyte proliferation by inhibiting the activity of this enzyme, rather than by non-specific effects. Aphidicolin, an inhibitor of alpha-polymerase, also prevented DNA strand break repair in conA-stimulated cells but, unlike MBA, did not prevent blast formation. DNA strand breaks accumulated in the presence of MBA at the same linear rate (300-400/h) in both resting and conA-treated cells. We and others had hypothesized that this accumulation was due to a continuous production of strand breaks in lymphocytes, leading to their accumulation in presence of repair inhibitors. However, incubation of the cells with aphidicolin at concentrations that inhibited repair did not result in any increase in strand breaks. The hypothesis of continuous cycling of breaks is incorrect; accumulation of breaks was due to some indirect effect of MBA, such as a possible disinhibition of an ADP-ribosylation-sensitive endonuclease described in other cell types. All of the early stages of lymphocyte proliferation, including blast transformation (but not DNA synthesis) require ADP ribosylation. Repair of DNA strand breaks is not a precondition for blast formation, though experiments involving the combined effects of MBA and aphidicolin showed that repair of the breaks is essential in order for the cells to replicate their DNA. Our data are consistent with a model suggesting that DNA strand breaks introduced into differentiated cells act as an additional safety-catch mechanism that restrains them from replicating their genetic material but not from undergoing the early stages of proliferation.
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PMID:Early nuclear events in lymphocyte proliferation. The role of DNA strand break repair and ADP ribosylation. 309 84

The diminution of NAD level in mouse thymus lymphocytes precedes their death under the effect of various genotoxic agents and manifests itself by the time of the onset of chromatin degradation. At the same time, in vitro, NAD does not influence the activity of micrococcus nuclease of Ca2+,Mg2+-dependent endonuclease from human spleen. Stimulation of protein poly(ADP-ribosylation) by exogenous NAD does not change the sensitivity of chromatin to micrococcus nuclease. In contrast to hepatocytes, in the thymus, no inhibition of Ca2+,Mg2+-endonuclease, resulting from ADP-ribosylation, occurs which may be due to low activity of ADP-ribosyl transferase in thymocytes. Incubation of thymus lymphocytes with benzamide prior to irradiation does not inhibit chromatin degradation. It is suggested that the decrease in the NAD level is one of the indications of the injury to thymocytes which is not related to the induction of their death. In contrast to thymocytes, the pretreatment of Ehrlich ascites tumor cells with benzamide produces a radiosensitizing effect.
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PMID:[Participation of the NAD-poly(ADP ribose) system in the degradation of chromatin in irradiated thymocytes]. 325 28

It no longer seems likely that DNA molecules in situ have a uniform conformation, represented by the classical B-form helix. For example, recent structural studies have shown that in certain conditions DNA can have a left-handed (so-called Z-form) helix, and have revealed extensive sequence-dependent variations of B-DNA helical parameters. Such sequence-dependent variations in DNA structure can be investigated in solution with reagents that bind to DNA in a conformation-dependent manner, and cut one or both strands of the double-helix at the site of binding, as, for example, has been shown for the endonuclease DNase I3. We describe here a simple way to endow a DNA-binding ligand with the ability to cleave DNA--labelling with 125I. The radiochemical damage associated with 125I decay induces a double-stranded DNA break. Using this technique we have shown that a sequence of four consecutive A X T base pairs is a necessary, but not sufficient, condition for strong binding to DNA of the bis-benzamide Hoechst 33258--presumably the other important factor is the conformation of the double-helix at the site of the (A/T)4 sequence. We suggest 125I-Hoechst 33258 may be a useful new probe of DNA structure.
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PMID:Use of an 125I-labelled DNA ligand to probe DNA structure. 618 59

Exposure of renal proximal tubular epithelial cells (LLC-PK1) to the nephrotoxicants 2-bromo-6-(glutathion-S-yl)hydroquinone, 2-bromo-3-(glutathion-S-yl)-hydroquinone, and 2-bromo-(diglutathion-S-yl)hydroquinone caused DNA fragmentation and cytotoxicity. Viability measured by lysosomal neutral red accumulation was the most sensitive parameter of cytotoxicity, and preceded toxicity determined by either the mitochondrial MTT assay or by measuring intracellular lactate dehydrogenase activity. DNA fragmentation was detected as early as 15 min after exposure to 2-bromo-6-(glutathion-S-yl)hydroquinone (100 microM), 2-bromo-3-(glutathion-S-yl)hydroquinone (200 microM), and 2-bromo-(diglutathion-S-yl)hydroquinone (400 microM) and prior to other indices of toxicity. The ability of the cells to repair DNA damage was evident by the decrease in the extent of single strand breaks following removal of 2-bromo-3-(glutathion-S-yl)hydroquinone from the incubation medium. Moreover, inhibition of poly(ADP-ribose)polymerase with 3-amino-benzamide (10 mM), following exposure of LLC-PK1 cells to 0.5 mM 2-bromo-6-(glutathion-S-yl)hydroquinone or 2-bromo-(diglutathion-S-yl)hydroquinone, decreased cytotoxicity, indicating that DNA repair processes, activated in response to DNA damage, exacerbate toxicity. Treatment with the endonuclease inhibitor, aurintricarboxylic acid did not decrease cytotoxicity. A decrease in the cytotoxicity caused by 2-bromo-6-(glutathion-S-yl)hydroquinone and 2-bromo-(diglutathion-S-yl)hydroquinone was observed when cells were incubated with catalase or pretreated with deferoxamine (10 mM). The data suggest a mechanism whereby the conjugates generate hydrogen peroxide, and the subsequent iron-catalyzed generation of hydroxyl radicals causes DNA fragmentation and cytotoxicity.
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PMID:Reactive oxygen species and DNA damage in 2-bromo-(glutathion-S-yl) hydroquinone-mediated cytotoxicity. 779 84

We have evaluated the influence of protein poly(ADP-ribosyl)ation in the relaxation of chromatin by exposing a rat liver polynucleosomal extract to micrococcal nuclease (MNase) digestion. The kinetic susceptibility of polynucleosomes to endonuclease digestion was determined as a function of the time of incubation as well as endonuclease concentration. To validate our assay, we also ran control experiments with protein-free calf thymus DNA as the opposite of polynucleosomal DNA. Rat liver chromatin was also incubated in the absence or presence of exogenously added 200 microM betaNAD(+), the poly(ADP-ribosyl)ation substrate, before MNase digestion. For incubations in the presence of betaNAD(+), the synthesis of polynucleosomal poly(ADP-ribose) was stopped with 1 mM benzamide. After addition of MNase, endonuclease digestion was blocked with EDTA to chelate the Mg(2+) ions needed for enzymatic activation, and the samples were subjected to electrophoresis through 1.5% agarose gels. As expected, a faster degradation of chromatin into oligonucleosomal DNA ladders was observed upon protein poly(ADP-ribosyl)ation when the chromatin extract was preincubated with 200 microM betaNAD(+). Thus, our results are consistent with the conclusion that the covalent poly(ADP-ribosyl)ation of polynucleosomal proteins favors a more "relaxed" or "open" structure, which renders chromatin more susceptible to MNase digestion.
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PMID:Polynucleosomal synthesis of poly(ADP-ribose) causes chromatin unfolding as determined by micrococcal nuclease digestion. 1565 42

Higher order chromatin degradation (HOCD) is a stepwise dismantling of the genome through the excision of chromatin loops and their oligomers at matrix attachment regions (MARs) during the early stages of programmed cell death. Although HOCD ultimately leads to the inactivation of the genome and cell death, a partial HOCD in cells receiving sublethal signals may result in the loss of genetic stability leading to neoplasia, degeneration, and aging. The present study was undertaken to determine the role of protein poly(ADP-ribosyl)ation in HOCD. Nuclei isolated from rat glioma C6 cells were able to carry poly(ADP-ribosyl)ation as assessed by the incorporation of (32)P-NAD(+) into TCA-insoluble fraction. Under the same experimental conditions, millimolar NAD(+) induced rapid HOCD in nuclei. However, while poly(ADP-ribosyl)ation was totally abrogated by specific inhibitor, benzamide, NAD(+)-induced HOCD was unaffected. Benzamide also failed to inhibit HOCD induced by H(2)O(2) exposure in intact cells. These results indicate that HOCD is not mediated through chromatin poly(ADP-ribosyl)ation, and that NAD(+) activates MAR-associated endonuclease or facilitates the access of the enzyme to DNA by other mechanisms. Furthermore, other nucleotides including NADP(+), ATP, UTP, GTP, and CTP were also found to induce HOCD in isolated nuclei indicating that HOCD is controlled by nucleotide-related ligands.
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PMID:Nucleotides induce higher order chromatin degradation. 1631 10

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