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 complete set of dA and T analogues designed for the study of protein DNA interactions has been prepared. These modified bases have been designed by considering the groups on the dA and T bases that are accessible to proteins when these bases are incorporated into double-helical B-DNA [Seeman, N. C., Rosenberg, J. M., & Rich, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 804-808]. Each of the positions on the two bases, having the potential to interact with proteins, have been subject to nondisruptive, conservative change. Typically a particular group (e.g., the 6-NH2 of dA or the 5-CH3 of T) has been replaced with a hydrogen atom. Occasionally keto groups (the 2- and 4-keto oxygen atoms of T) have been replaced with sulfur. The base set has been incorporated into the self-complementary dodecamer d(GACGATATCGTC) at the central d(ATAT) sequence. Melting temperature determination shows that the modified bases do not destabilize the double helix. Additionally, circular dichroism spectroscopy shows that almost all the altered bases have very little effect on overall oligodeoxynucleotide conformation and that most of the modified oligomers have a B-DNA type structure. d(GATATC) is the recognition sequence for the EcoRV restriction modification system. Initial rate measurements (at a single oligodeoxynucleotide concentration of 20 microM) have been carried out with both the EcoRV restriction endonuclease and modification methylase. This has enabled a preliminary identification of the groups of the dA and T bases within the d(GATATC) sequence that make important contacts to both proteins.
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PMID:Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase. 227 27

Mitomycin C and certain analogues alkylate DNA with their C-1 position and cross-link it by a second alkylation involving C-10. We now show that monoalkylation by C-10 (carbamate group) can occur for mitosene analogues that have no reactive C-1 functionality. Sodium dithionite reduction of 2,7-diaminomitosene or 2,7-diamino-1-hydroxymitosene in the presence of calf thymus DNA resulted in alkylation of the DNA to the extent of one molecule per 14 and 11 bases, respectively, although no covalent binding was observed on catalytic reduction. Reduction of each of these mitosenes by sodium dithionite in the presence of 2'-deoxyguanosine gave monoalkylation on the 2-amino group of this nucleotide. The 2,7-diaminomitosenes inhibited L-1210 leukemia cell colony formation in vitro at concentrations 3-4-fold greater (less potent) than mitomycin C. DNA single-strand breaks were also produced by each mitosene, but these lesions did not correlate with cytotoxicity and were less prominent than breaks produced by another monofunctional alkylating agent, methyl methanesulfonate. Mitosene-induced DNA strand breaks are probably due to excission-repair endonuclease activity and not from oxygen free radicals produced by redox cycling of the quinone moiety. There was no evidence of DNA-DNA cross-links by either 2,7-diaminomitosene.
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PMID:Alkylation of DNA by C-10 of 2,7-diaminomitosene. 229 22

DNA single-strand breaks (ssb) induced by N-ethyl-N-nitrosourea (ENU) in CHO cells are quickly resealed within 10 min after treatment. This rapid repair kinetics is not explained by the rate of base excision repair which removes the main ethyl products with a half-life in the order of hours. We have explored the potential use of methoxyamine (MX), a chemical that reacts at neutral pH with AP sites in DNA in vitro, to clarify the origin of ENU-induced ssb. The presence of 50 mM MX during cell treatment with diethyl sulfate (DES) caused selective inhibition of the repair of AP sites generated during base excision repair and inhibited alkaline cleavage at these sites. The treatment of CHO cells with ENU in the presence of MX clearly showed that the burst of ssb observed immediately after treatment was due to AP site formation. Plasmid DNA treated in vitro with ENU did not present AP endonuclease-sensitive sites; therefore, the AP sites produced in CHO cells by ENU treatment are not due to the chemical hydrolysis of a very unstable ethyl adduct but rather are intermediates of an as yet undefined enzymatic pathway. This process occurs specifically after treatment with SN1-type ethylating agents (ENU and N-ethyl-N'-nitro-N-nitrosoguanidine) suggesting an association between this phenomenon and DNA-oxygen alkylation. We suggest that these breaks are generated by a mechanism of O6-ethylguanine processing without removal of the modified base.
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PMID:Evidence for AP site formation related to DNA-oxygen alkylation in CHO cells treated with ethylating agents. 236 94

Agents that act via oxygen-derived free radicals form DNA strand breaks with fragmented sugar residues that block DNA repair synthesis. Using a synthetic DNA substrate with a single type of sugar fragment, 3'-phosphoglycolaldehyde esters, we show that in Escherichia coli extracts the only EDTA-resistant diesterase for these damages depends on the bacterial nfo (endonuclease IV) gene. Endonuclease IV was purified to physical homogeneity (Mr = 31,000) from an E. coli strain carrying the cloned nfo gene and in which the enzyme had been induced with paraquat. Although heat-stable and routinely assayed in the presence of EDTA, endonuclease IV was inactivated in the absence of substrate at 23-50 degrees C by either EDTA or 1,10-phenanthroline, suggesting the presence of an essential metal tightly bound to the protein. Purified endonuclease IV released phosphoglycolaldehyde, phosphate, and intact deoxyribose 5-phosphate from the 3'-end of DNA, all with apparent Km of 5-10 nM. The optimal KCl or NaCl concentration for 3'-phosphoglycolaldehyde release was 50-100 mM. The purified enzyme had endonuclease activity against partially depurinated DNA but lacked significant nonspecific nuclease activities. Endonuclease IV also activated H2O2-damaged DNA for repair synthesis by DNA polymerase I. Thus, endonuclease IV can act on a variety of oxidative damages in DNA, consistent with a role for the enzyme in combating free-radical toxicity.
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PMID:Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. 245 10

Myeloperoxidase (MPO) is a critical component in the oxygen-dependent microbicidal activity of neutrophils. Hereditary deficiency of MPO occurs commonly, but its genetic basis has not been determined. Previously we have reported the presence of an 89-kilodalton protein, likely pro-MPO, in normal and MPO-deficient neutrophils and hypothesized that the absence of peroxidase activity in neutrophils from affected subjects was the result of defective posttranslational processing of pro-MPO. In this study we analyzed nucleic acids from three completely and two partially MPO-deficient individuals by using a cDNA probe for MPO. The affected individuals studied are unrelated to one another. Neutrophils from all affected subjects lacked mature MPO subunits; however, a monospecific antibody for MPO identified in these cells a high-molecular weight protein that is the same size as pro-MPO. Northern blots demonstrated that the amount and size of RNA (3.3 kilobases [kb]) in a completely deficient subject was normal. BglII digests of genomic DNA from control individuals (n = 14) contained three fragments that hybridized with cDNA for MPO under very stringent conditions. In contrast, BglII digests of genomic DNA from completely MPO-deficient individuals contained an extra fragment of 2.1 kb that was not present in DNA from controls. In addition, two different endonuclease digest patterns were found in MPO-deficient subjects who were biochemically and phenotypically identical. We conclude from these studies that (a) hereditary MPO deficiency is not associated with a major deletion or rearrangement of the MPO gene; (b) myeloid precursors in an MPO-deficient individual contain normal amounts of an mRNA that is the same size as that for MPO in normal individuals; and (c) the genetic basis for MPO deficiency may be heterogeneous, with at least two genotypes generating the same phenotype. These findings are consistent with the hypothesis that the genetic defect in MPO deficiency results in synthesis of a modified pro-MPO that undergoes defective posttranslational processing.
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PMID:Aberrant restriction endonuclease digests of DNA from subjects with hereditary myeloperoxidase deficiency. 246 38

DNA photosensitization by several furocoumarins (including 3-carbethoxypsoralen (3-CPs), 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin was investigated by using DNA sequencing methodology. 3-CPs induces photo-oxidation of guanine residues leading to alkali-labile sites in DNA (revealed by hot piperidine), whereas 8-MOP, 5-MOP and angelicin do not. There is a preferential photo-oxidation of G when located on the 5' side of GG doublets, likely to reflect a better accessibility of the G moiety in such a context. Mechanisms operating via both radicals (type I) and singlet oxygen (type II) are involved in the photo-oxidation of G residues by 3-CPs. Photo-oxidized G residues are produced independently of the formation of photoadducts, and scavengers of singlet oxygen or radicals do not inhibit photobinding of 3-CPs to DNA. This leads us to propose that covalent photoadducts arise from the intercalated excited sensitizer molecules, whereas G photo-oxidations are produced either by electron transfer reactions involving bound 3-CPs or by energy transfer to molecular oxygen, thereby producing singlet oxygen that subsequently reacts with guanine bases. Quantification of both types of DNA lesions indicated that in vitro photo-oxidized G residues are produced in DNA by 3-CPs plus ultraviolet light at least to the same extent as photoadducts, under our conditions. A calf thymus redoxyendonuclease, equivalent to the endonuclease III of Escherichia coli, specific for oxidative DNA damages, recognizes and cleaves DNA at sites of photo-oxidized G residues. The extent of the cleavage by this enzyme was close to that observed by hot piperidine and followed the amount of photo-oxidized G residues produced when the lifetime of excited oxygen species is modified. The redoxyendonuclease did not incise DNA treated with 8-MOP, 5-MOP or angelicin plus ultraviolet light. The exonuclease III and endonuclease IV of E. coli also involved in the repair of oxidative DNA damage, convert the replicative form I of 3-CPs-treated DNA to replicative form II. This suggests that the lesions recognized by these enzymes are apurinic-like lesions. In view of the low toxicity and mutagenicity of 3-CPs, DNA photo-oxidation products induced by the photodynamic effect of 3-CPs are likely to be efficiently taken care of by the DNA repair system(s). It is clear that 3-CPs photo-induces several classes of DNA damage, including oxidative damage.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Oxidative DNA damage photo-induced by 3-carbethoxypsoralen and other furocoumarins. Mechanisms of photo-oxidation and recognition by repair enzymes. 247 51

An integrated view of the processes which most likely play a critical role in the aging process at the cellular level is proposed. Cells are continuously exposed to a variety of internal and external stressors, potentially dangerous for the maintenance of the functional integrity of the cell (UV and gamma radiation, heat, oxygen free radicals, glucose, bacteria, viruses). In the course of evolution a number of mechanisms [DNA repair, production of heat shock and other stress proteins, enzymatic and non-enzymatic antioxidant defence systems, poly(ADP-ribose) polymerase activation] have emerged which allow the cell to cope with such a variety of potentially harmful agents. These mechanisms are in fact interconnected and constitute a network of cellular defence systems. It is suggested that they play a physiological role, being involved in the control of gene expression. A failure of these mechanisms does not allow the cell to maintain homeostasis and has profound consequences as far as two of the major programs of the cell are concerned, i.e. cell proliferation and cell death. Recent data suggesting that these are two physiologically active phenomena tightly linked and regulated are examined. Thus, activation of cell cycle related genes and active inhibition of suicide genes appear to be a part of an integrated process. Conversely, deprivation of growth factors seems able to induce an active process of programmed cell death characterized by Ca++,Mg+(+)-dependent endonuclease activity and DNA fragmentation (apoptosis). Similar phenomena have been shown to accompany the terminal differentiation process in several cellular systems. The understanding of the factors which favour or prevent cell death (a phenomenon which has been recognized as one of the most important in fetal development and morphogenesis) will help to unravel and eventually to manipulate the aging process. In an evolutionary perspective, cell senescence appears to be the price paid to avoid unlimited capability of proliferation, i.e. cell transformation and cancer.
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PMID:Cell proliferation, cell death and aging. 248 97

Endonuclease protection or "footprinting" analysis is a powerful technique for identifying the nucleotides involved in a protein-DNA interaction. DNase I is the most often employed endonucleolytic agent; however, this endonuclease does not exhibit the true nonsequence-specific cleavage desired for this type of analysis. Methidiumpropyl-EDTA (MPE) is a synthetic DNA intercalator that cleaves DNA in the presence of ferrous ion and oxygen. Cleavage by MPE exhibits no sequence specificity, a characteristic that makes this reagent better suited for protection analysis. Here we report a generally applicable technique for MPE protection (or "footprinting") analysis of specific DNA-protein complexes from a crude nuclear extract. We have used this method to identify the nucleotides of the immunoglobulin (Ig) heavy chain promoter region that are involved in complex formation with a protein that binds the octameric sequence ATGCAAAT, and we compare our results to those obtained previously using DNase I.
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PMID:Protection analysis (or "footprinting") of specific protein-DNA complexes in crude nuclear extracts using methidiumpropyl-EDTA-iron (II). 251 95

The specific recognition of various DNA modifications by repair enzymes is exploited for the analysis of DNA damage induced by visible light in the presence of methylene blue in Salmonella typhimurium. The relative frequencies of various endonuclease-sensitive sites and strand breaks are determined in the plasmid pAQ1 of the treated bacteria and are compared with those observed after exposure of isolated DNA to various conditions. This comparison of damage profiles indicates that the cellular DNA damage by illumination in the presence of methylene blue is caused predominantly by the direct action of singlet oxygen. Indirect mechanisms, e.g. involving a generation of superoxide and hydroxyl radicals or the activation of cellular nucleases, do not contribute very much. The damage is dominated by base modifications. These are subject to an efficient repair that is not mediated by uvrABC proteins and therefore most probably involves recognition by specific glycosylases. Revertant frequencies observed under these conditions in the strains TA1535, TA100, TA2638 and TA104 indicate a pronounced mutagenicity of the lesions induced. On the other hand, the DNA damage does not contribute significantly to the cytotoxicity caused by the treatment as an excision repair deficiency (uvrB) has no influence on cell killing.
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PMID:Singlet oxygen as an ultimately reactive species in Salmonella typhimurium DNA damage induced by methylene blue/visible light. 268 Jan 44

A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe [Brown, E. R., Coker, G. T., III, & O'Malley, K. L. (1987) Biochemistry 26, 5208-5212]. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping. Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase [DiLella, A. G., Kwok, S. C. M., Ledley, F. D., Marvit, J., & Woo, S. L. C. (1986) Biochemistry 25, 743-749] genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence.
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PMID:Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs. 289 28


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