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 effect of glycerol on the specificity of DNA cleavage by the restriction endonuclease BamHI has been examined. In addition to the canonical G decreases from G-A-T-C-C site, BamHI cuts DNA at several sites that we have named noncanonical BamHI.1 sites. The number of BamHI.1 sites in simian virus 40 and pBR322 was determined to be 13 for each DNA. Cutting sites determined by DNA sequence analysis include G decreases from G-A-A-C-C, G decreases from G-C-T-C-C, G decreases from G-G-T-C-C, and G-A-A-T-C-C with the complementary strand sequence assignments of G-G-T-T-C-C, G-G-A-G-C-C, G-G-A-C-C-C, and G-G-A-T-T-C. The relaxation in specificity was related to hydrogen bond acceptor and donor sites in the recognition sequence, in an attempt to generate a model of BamHI recognition of cognate sites in DNA.
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PMID:Sequence-specific endonuclease BamHI: relaxation of sequence recognition. 628 22

A mitochondrial endonuclease from Saccharomyces cerevisiae was previously shown to cut both strands of native DNA at opposite or nearby sites. The present studied demonstrate that the endonuclease activity is dependent on the strength of the hydrogen bonds between the DNA strands; the activity was measured at different ionic strengths, with substrates of different base compositions and also with DNA in which the double helix has been locally destabilized by ultraviolet irradiation, by depurination, and by single-stranded nicks. The activity is 30% greater with mitochondrial DNA (mt-DNA) than with nuclear DNA. At 0.08 ionic strength, the relative activities with double-stranded polydeoxyribonucleotides are 2.4:1:0.6 for poly(dA).poly(dU) : poly(dA).poly(dT) : poly(dG). poly(dC). Increasing ionic strength decreases similarly the activity with poly(dA).poly(dU) and poly(dA).poly(dT), but has little effect with poly(dG).poly(dC). The greater activity with poly(dA).poly(dU) than with poly(dA).poly(dT) was confirmed with nick-translated mt-DNA and with DNA synthesized in isolated mitochondria using [3H]TTP and [3H]dUTP in both cases. The endonuclease cuts modified DNA preferentially in the thymine dimer regions, at the apurinic sites, and at sites opposite to nicks. The possible involvement of this endonuclease in the degradation of mitochondrial DNA during "petite" induction is discussed.
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PMID:Some properties of a mitochondrial endonuclease from yeast. 628 8

Under standard conditions, Eco RI endonuclease uniquely recognizes the inverted repeat GAATTC. However, this specificity breaks down under non-standard conditions into what has been termed Eco RI* specificity, wherein many other sequences are recognized. We show here that the hydrolysis rates at all known Eco RI* sites can be summarized by the hierarchies: G much greater than A greater than T much greater than C at the first position, A much greater than [G,C] much greater than T at the second and third position, and the corresponding complements at the last three positions. This is consistent with a recognition model which assumes that there are two specific hydrogen bonds per base pair under standard conditions. One or more of these are randomly replaced by water under Eco RI* conditions and the position of a sequence within the appropriate hierarchy is primarily determined by the number of retained hydrogen bonds. These retained hydrogen bonds are common recognition features that can be identified by examining the DNA. The recognition points thereby identified for Eco RI all fall within the major groove of the DNA.
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PMID:Eco RI* specificity and hydrogen bonding. 629 67

Eighteen EMS-induced mutant strains of S. cerevisiae with increased sensitivity to petite induction by sulfanilamide-aminopterin treatment have been isolated. Four of these strains demonstrated a concomitant increase in sensitivity to the petite-inducing effects of u.v. irradiation and of growth at an elevated temperature. Of these, two were shown to be a consequence of recessive nuclear mutations at one gene (sas1-1 and sas1-2). Expression of the two remaining mutations was too low to permit genetic analysis. All three petite-inducing treatments used in this study are known to reduce the hydrogen bond strength between the two strands of double stranded DNA, and the existence of multiple sensitivity is discussed in terms of an alteration of a mitochondrial endonuclease which acts preferentially at sites of reduced attraction.
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PMID:Nuclear mutations in Saccharomyces cerevisiae conferring multiple sensitivity to petite-inducing treatments. 676 51

A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-stranded-specific ribonuclease V1 from the venom of the cobra Naja naja oxiana. 32p-end-labeled RNA is first partially digested with double-strand-specific V1 nuclease under near physiological conditions, and the resultant fragments are than electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel run in 90% formamide. After autoradiography, the base-paired nucleotides are definitively located by comparing V1 generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNAPhe an E, coli tRNAfMet of known three-dimensional structure, we find V1 nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V1 will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNAPhe cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.
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PMID:Mapping tRNA structure in solution using double-strand-specific ribonuclease V1 from cobra venom. 703 4

We have cloned mouse and human cDNAs for a multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3',5' exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. To investigate the biological role of APEX nuclease, sense or antisense APEX RNA was stably expressed at a high level in cultured rat glioma cells by introducing plasmids (pABWN-HAPX1F for expression of sense RNA or pABWN-HAPX2R for expression of antisense RNA) constructed from the human APEX cDNA and an expression vector pABWN. Multiple copies of the construct were integrated into the glioma cells transfected with pABWN-HAPX1F or pABWN-HAPX2R. These transfectants showed markedly high expression of RNA hybridizable to human APEX cDNA, indicating the expression of the sense or antisense RNA. Activity blotting analyses of salt extracts of these transfectants showed that the sense RNA-expressed cells had higher AP endonuclease activity and that the antisense RNA-expressed cells had extremely lower AP endonuclease activity than the control cells. The APEX nuclease-depressed glioma cells became more sensitive to methyl methanesulfonate and hydrogen peroxide than the control cells or the APEX nuclease-overexpressed cells. The results indicate that APEX nuclease plays an important role in repair of DNA damage caused by these genotoxic agents. The present stable expression systems for the sense and antisense APEX RNAs should be useful for analyzing the biological functions such as an antimutagenic function of the enzyme.
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PMID:Stable expression in rat glioma cells of sense and antisense nucleic acids to a human multifunctional DNA repair enzyme, APEX nuclease. 751 11

Bovine pancreatic deoxyribonuclease I is an endonuclease of low specificity that interacts with the minor groove of DNA. Two amino acids, R41 and Y76, completely fill this groove, with R41 hydrogen bonding to the O2/N3 positions of pyrimidines and purines, and Y76 contacting a deoxyribose via an unusual hydrophobic "stacking" interaction. The roles of these amino acids in phosphodiester bond cleavage and in DNA hydrolysis selectivity have been studied by site-directed mutagenesis. Alterations have been made that are either conservative (R41K, Y76F) or more drastic (R41A, R41G, Y76A, Y76G). The surface loop (residues 73 to 76) that contains Y76 has also been deleted. Several double mutants in which both R41 and Y76 have been altered have also been prepared. The integrity of the catalytic site of the mutants has been investigated using the small, non-DNA, chromophoric substrate deoxythymidine-3',5'-di-(p-nitrophenyl)-phosphate. Hydrolysis of this compound was hardly changed, even by the most extreme alterations to R41 and Y76. In contrast, all the mutants bound DNA about ten times more weakly than the wild-type and, with the exception of R41K and Y76F, hydrolysed DNA much more slowly. This suggests that changes to R41 and Y76 have little effect on catalytic amino acids at the hydrolysis site, but are required to bind DNA and, more importantly, to correctly position the scissile phosphate for efficient hydrolysis. The selectivity of DNA hydrolysis for all the mutants has been tested using the 160 base-pair Escherichia coli Tyr T promoter DNA fragment. Very small differences were seen in global hydrolysis selectivity when either amino acid was altered. However, changes to R41 resulted in some differences to local cutting specificity that could be explained by the role of this amino acid in hydrogen bonding to particular bases relative to the scissile phosphate.
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PMID:The roles of arginine 41 and tyrosine 76 in the coupling of DNA recognition to phosphodiester bond cleavage by DNase I: a study using site-directed mutagenesis. 765 Jul 37

The ribozyme derived from the intron of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides. The total transition-state stabilization by the ribozyme, encompassing the binding and chemical steps, is 4.8 kcal/mol greater with a single ribose at the cleavage site relative to the all-deoxyribose substrate. Here we show that this effect is specific to the chemical transition state, with a contribution of only approximately 0.7 kcal/mol toward binding. Substrates with a series of 2'-substituents, -OH(ribo), -F2 (2',2'-difluoro-2'-deoxyribo), F(2'-fluoro-2'-deoxyribo), and -H(deoxyribo), follow a linear free energy relationship between the rate of the chemical step of the ribozyme-catalyzed reaction and the pK(a) of the leaving group, with slope beta leaving group approximately -0.8. Because proton donation to the 3'-oxygen atom from a general acid of the ribozyme would be expected to render the rate insensitive to the pK(a) of the leaving group, it is suggested that this ribozyme does not employ general acid catalysis. The 2'-OCH3 (2'-methoxy-2'-deoxyribo) substituent does not follow this correlation, apparently due to steric hindrance within the active site. The rate of cleavage of the 2'-substituted substrates by the ribozyme follows the order 2'-F2 > -F > -H, suggestive of an inductive effect, i.e., acceleration of the reaction by electron-withdrawing groups. The 2'-OH group provides the largest transition-state stabilization. Because of uncertainty in the relative effect of the 2'-OH and 2'-H substituents on the pK(a) of the neighboring 3'-oxygen leaving group, we do not discount the possibility of interactions between the 2'-hydroxyl group and the ribozyme that further enhance reactivity. Nevertheless, the 2'-OH effect can be explained at least partially by an intramolecular hydrogen bond to an incipient oxyanion at the neighboring 3'-position. This oxyanion is forming as the phosphodiester bond is breaking, explaining why the stabilization is specific to the transition state. Analogous differential hydrogen bonding might be widely used by enzymes to achieve selective transition-state stabilization.
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PMID:The importance of being ribose at the cleavage site in the Tetrahymena ribozyme reaction. 768 73

Ionizing radiation produces a variety of DNA damage through active oxygen species such as the superoxide radical (O2.-), the hydroxyl radical (OH.), and hydrogen peroxide (H2O2). The removal of alkylation-induced apurinic (AP) sites and 3'-blocking deoxyribose fragments by exonuclease III (xth) and endonuclease IV (nfo) has been well demonstrated in E. coli. Very little information on the repair of radiation-induced DNA damage by human apurinic endonuclease is available. We examined the biological roles of the human AP endonuclease in the repair of radiation-induced DNA damage. An expression vector was constructed with human APE cDNA and transformed into radiation-sensitive E. coli mutants (xth- and nfo-). The radiation cytotoxicity was assayed by cell survival curves. Expression of human AP endonuclease in E. coli confirmed that AP endonuclease could complement exonuclease III functionally to diminish radiation cytotoxicity. In contrast, AP endonuclease was not able to increase resistance to H2O2, owing to a poor 3'-termini repair. We also tested whether AP endonuclease is a limiting factor for radiation cytotoxicity by using a plasmid nicking assay. Cell extracts from mutant cells with or without AP endonuclease expression were added to irradiated supercoiled plasmid DNA. The inability to convert supercoiled plasmid DNA to relaxed or linear forms suggested that there were large accumulations of AP sites in the mutant cell extracts. The AP endonuclease activities estimated from the plasmid nicking assays are 20-fold lower in the cell extracts of AP endonuclease-deficient mutant than in AP endonuclease-expressing cells. Therefore, AP endonuclease is a limiting step of base excision repair for the radiation-sensitive E. coli mutant, BW528. Our results conclude that AP endonuclease is responsible for the removal of AP sites from gamma-radiation-induced base damage in E. coli.
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PMID:Reduction of radiation cytotoxicity by human apurinic endonuclease in a radiation-sensitive Escherichia coli mutant. 769 Sep 78

Drosophila Rrp1 protein has four tightly associated enzymatic activities: DNA strand transfer, ssDNA renaturation, dsDNA 3'-exonuclease and apurinic/apyrimidinic (AP) endonuclease. The carboxy-terminal region of Rrp1 is homologous to Escherichia coli exonuclease III and several eukaryotic AP endonucleases. All members of this protein family cleave abasic sites. Rrp1 protein was expressed under the control of the E. coli RNA polymerase tac promoter (pRrp1-tac) in two repair deficient E. coli strains (BW528 and LG101) lacking both exonuclease III (xth) and endonuclease IV (nfo). Rrp1 confers resistance to killing by oxidative, antitumor and alkylating agents that damage DNA (hydrogen peroxide, t-butylhydroperoxide, bleomycin, methyl methanesulfonate, and mitomycin C). Complementation of the repair deficiency by Rrp1 provides up to a two log increase in survival and requires the C-terminal nuclease region of Rrp1, but not its N-terminal region. The AP endonuclease activity in extracts from the repair deficient strain LG101 is increased up to 12-fold when the strain contains pRrp1-tac. These results indicate that pRrp1-tac directs the synthesis of active enzyme, and that the nuclease activities of Rrp1 are likely to be the cause of the increased resistance to DNA damage of the mutant cells.
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PMID:Drosophila Rrp1 complements E. coli xth nfo mutants: protection against both oxidative and alkylation-induced DNA damage. 769 34


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