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 PI-SceI endonuclease from yeast belongs to a protein family whose members contain two conserved dodecapeptide motifs within their primary sequences. The function of two acidic residues within these motifs, Asp218 and Asp326, was examined by substituting alanine, asparagine, and glutamic acid residues at these positions. All of the purified mutant proteins bind to the PI-SceI recognition site with the same affinity and specificity as the wild-type enzyme. By contrast, substituting alanine or asparagine amino acids at the two positions completely eliminates strand cleavage of substrate DNA, whereas substitution with glutamic acid markedly reduces the cleavage activity. Experiments using nicked substrates demonstrate that the wild-type enzyme shows no strand preference during cleavage. These results are consistent with a model in which both acidic residues are part of a single catalytic center that cleaves both DNA strands. Furthermore, substrate binding by wild-type PI-SceI stimulates hydroxyl radical or hydroxide ion attack at the cleavage site while binding by the alanine-substituted proteins either stimulates this attack significantly less or protects the DNA at this position. These finding are discussed in terms of possible reaction mechanisms for PI-SceI-mediated endonucleolytic cleavage.
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PMID:Substitutions in conserved dodecapeptide motifs that uncouple the DNA binding and DNA cleavage activities of PI-SceI endonuclease. 789 Jul 14

Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antisense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E. coli carrying the aadA gene construct, and transformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.
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PMID:Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease. 790 39

Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C-terminal domain likely to possess a DNA-binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate.
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PMID:A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues. 873 63

HAP1 is a divalent cation-dependent endonuclease from human cells with specificity for apurinic/apyrimidinic (AP) sites in DNA. Extraction of the essential metal ion from purified HAP1 stabilized its binding to an oligonucleotide containing a single AP site, permitting AP site binding studies to be undertaken using gel retardation assays. Binding of HAP1 to such an oligonucleotide was dependent upon the presence of an AP site. Previous structural and modelling studies have suggested a role for Asn212 (Asn153 in exonuclease III, the bacterial homologue of HAP1) in substrate recognition. Substitution of alanine for Asn212 abolished the AP endonuclease activity of purified recombinant HAP1 protein. More conservative substitutions of aspartate or glutamine for Asn212 still led to a reduction in specific activity of at least 300-fold. Moreover, none of the three Asn212 substitution mutants of HAP1 possessed detectable AP site binding activity in vitro. This study indicates that chelation of the active site metal ion in HAP1 stabilizes the complex of the protein with AP sites and identifies an active site asparagine residue as an important component of AP site recognition by the HAP1 protein.
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PMID:Asparagine 212 is essential for abasic site recognition by the human DNA repair endonuclease HAP1. 893 75

We studied the DNA sequence of the entire coding region of ERCC1 gene, in five cell lines established from human ovarian cancer (A2780, A2780/CP70, MCAS, OVCAR-3, SK-OV-3), 29 human ovarian cancer tumor tissue specimens, one human T-lymphocyte cell line (H9), and non-malignant human ovary tissue (NHO). Samples were assayed by PCR-SSCP and DNA sequence analyses. A silent mutation at codon 118 (site for restriction endonuclease MaeII) in exon 4 of the gene was detected in MCAS, OVCAR-3 and SK-OV-3 cells, and NHO. This mutation was a C-->T transition, that codes for the same amino acid: asparagine. This transition converts a common codon usage (AAC) to an infrequent codon usage (AAT), whereas frequency of use is reduced two-fold. This base change was associated with a detectable band shift on SSCP analysis. For the 29 ovarian cancer specimens, the same base change was observed in 15 tumor samples and was associated with the same band shift in exon 4. Cells and tumor tissue specimens that did not contain the C-->T transition, did not show the band shift in exon 4. Our data suggest that this alteration at codon 118 within the ERCC1 gene, may exist in platinum-sensitive and platinum-resistant ovarian cancer tissues.
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PMID:A nucleotide polymorphism in ERCC1 in human ovarian cancer cell lines and tumor tissues. 936 Jun 34

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.
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PMID:Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate. 968 58

We investigated the interaction dynamics of human abasic endonuclease, the Ape1 protein (also called Ref1, Hap1, or Apex), with its DNA substrate and incised product using electrophoretic assays and site-specific amino acid substitutions. Changing aspartate 283 to alanine (D283A) left 10% residual activity, contrary to a previous report, but complementation of repair-deficient bacteria by the D283A Ape1 protein was consistent with its activity in vitro. The D308A, D283/D308A double mutant, and histidine 309 to asparagine proteins had 22, 1, and approximately 0. 02% of wild-type Ape1 activity, respectively. Despite this range of enzymatic activities, all the mutant proteins had near-wild-type binding affinity specific for DNA containing a synthetic abasic site. Thus, substrate recognition and cleavage are genetically separable steps. Both the wild-type and mutant Ape1 proteins bound strongly to the enzyme incision product, an incised abasic site, which suggested that Ape1 might exhibit product inhibition. The use of human DNA polymerase beta to increase Ape1 activity by eliminating the incision product supports this conclusion. Notably, the complexes of the D283A, D308A, and D283A/D308A double mutant proteins with both intact and incised abasic DNA were significantly more stable than complexes containing wild-type Ape1, which may contribute to the lower turnover numbers of the mutant enzymes. Wild-type Ape1 protein bound tightly to DNA containing a one-nucleotide gap but not to DNA with a nick, consistent with the proposal that substrate recognition by Ape1 involves a space bracketed by duplex DNA, rather than mere flexibility of the DNA.
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PMID:Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product. 980 98

Amino acid residues Asn116 and Ser118 of the restriction endonuclease BamHI make several sequence-specific and water-bridged contacts to the DNA bases. An in vivo selection was used to isolate BamHI variants at position 116, 118 and 122 which maintained sequence specificity to GGATCC sites. Here, the variants N116H, N116H/S118G and S118G were purified and characterized. The variants N116H and N116H/S118G were found to have lost their ability to cleave unmethylated GGATCC sequences by more than two orders of magnitude, while maintaining nearly wild-type levels of activity on the N6-methyladenine-containing sequence, GGmATCC. In contrast, wild-type BamHI and variant S118G have only a three- to fourfold lower activity on unmethylated GGATCC sequences compared with GGmATCC sequences. The N116 to H116 mutation has effectively altered the specificity of BamHI from an endonuclease which recognizes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GGmATCC. The N116H change of specificity is due to the lowered binding affinity for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact between the imidazole group of histidine and the N6-methyl group of adenine.
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PMID:A mutant of BamHI restriction endonuclease which requires N6-methyladenine for cleavage. 991 94

A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.
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PMID:A novel endonuclease mechanism directly visualized for I-PpoI. 1058 47

The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro. We have carried out a mutational analysis of these presumptive Mg(2+) ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed.
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PMID:Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI. 1112 16


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