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)

Endonuclease I is a 149 amino acid protein of bacteriophage T7 that is a Holliday junction-resolving enzyme, i.e. a four-way junction-selective nuclease. We have performed a systematic mutagenesis study of this protein, whereby all acidic amino acids have been individually replaced by other residues, mainly alanine. Out of 21 acidic residues, five (Glu20, Glu35, Glu65, Asp55 and Asp74) are essential. Replacement of these residues by other amino acids leads to a protein that is inactive in the cleavage of DNA junctions, but which nevertheless binds selectively to DNA junctions. The remaining 16 acidic residues can be replaced without loss of activity. The five critical amino acids are located within one section of the primary sequence. It is rather likely that their function is to bind one or more metal ions that coordinate the water molecule that brings about hydrolysis of the phosphodiester bond. We have also constructed a mutant of endonuclease I that lacks nine amino acids (six of which are arginine or lysine) at the C-terminus. Unlike the acidic point mutants, the C-terminal truncation is unable to bind to DNA junctions. It is therefore likely that the basic C-terminus is an important element in binding to the DNA junction.
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PMID:Catalytic and binding mutants of the junction-resolving enzyme endonuclease I of bacteriophage t7: role of acidic residues. 986 97

The PvuII restriction endonuclease is a homodimer that recognizes and cleaves the DNA sequence 5'-CAGCTG-3' in double-stranded DNA, and the structure of this enzyme has been reported. In the wild-type enzyme, Asp34 interacts with the internal guanine of the recognition sequence on the minor groove side. The Asp34 codon was altered to specify Gly (D34G), and in vitro studies have revealed that the D34G protein has lost binding specificity for the central G.C base-pairs, and that it cuts the canonical sequence with 10(-4)-fold reduced activity as compared to the wild-type enzyme. We have now determined the structure at 1.59 A resolution of the D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide containing the cognate sequence. The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes. First, the sugar moiety of the internal guanine changes from a C2'-endo to C3'-endo pucker while that of the 3' guanine changes from C3'-endo to C2'-endo pucker. Second, the axial rise between the internal G.C base-pairs is reduced while that between the G.C and flanking base-pairs is expanded. Third, two distinct monomeric active sites are observed that we refer to as being "primed" and "unprimed" for phosphodiester bond cleavage. The primed and unprimed sites differ in the conformation of the Asp58 side-chain, and in the absence from unprimed sites of four networked water molecules. These water molecules, present in the primed site, have been implicated in the catalytic mechanism of this and other endonucleases; some of them can be replaced by the Mg2+ necessary for cleavage. Taken together, these structural changes imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. This provides some insight into how recognition of the specific DNA sequence is linked to catalysis by the highly specific restriction endonucleases, and reveals one way in which the structural conformation of the DNA is modulated coordinately with that of the PvuII protein.
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PMID:Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis. 987 66

Characteristic steps in the course of cellular apoptosis are the induction of chromatin condensation and cleavage of the DNA, leading to the formation of oligomers of nucleosomes. Since the H1 histones represent functional elements that are essential for the generation of highly condensed chromatin structures, we analysed the total cellular H1 histones of five leukaemic and three solid human tumour cell lines, comparing the H1 pattern of exponentially growing cells with that of apoptotic cells. For the induction of apoptosis, cell lines were treated with the water-soluble camptothecin derivative, topotecan (a topoisomerase I inhibitor), or with an apoptosis-inducing monoclonal anti-CD95 (Fas/APO-1) antibody. Total histone H1 proteins were isolated by extraction with 5% perchloric acid and were analysed by means of capillary zone electrophoresis (CZE) separation. The identities of the peaks representing different histone H1 subtypes on CZE electropherograms were confirmed by analysis of preparations (recombinant proteins purified from transformed yeast used as internal standards) mixed with each of the subtypes respectively. The progress of topotecan- or anti-CD95-induced cell death was monitored by flow cytometry analysis and also by agarose electrophoresis of fragmented DNA. During early apoptosis of three of these cell lines, we observed the induction of internucleosomal DNA cleavage and, simultaneously, a typical change in the histone H1 protein pattern, leading to an increase in the relative amounts of histone subtypes H1.4 and H1.5. Upon apoptosis induction, these changes were only observed in correlation with the occurrence of DNA fragmentation, thus possibly reflecting a prerequisite for DNA accessibility and/or endonuclease activity.
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PMID:Changes in the protein pattern of H1 histones associated with apoptotic DNA fragmentation. 988 31

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 new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.
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PMID:A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase. 1019 86

The roles of divalent metal ions in DNA cleavage by the EcoRV endonuclease were studied by using Co2+ or Mn2+ as substitutes for the natural cofactor Mg2+. In steady-state experiments with a 12 bp oligonucleotide substrate, Co2+ yielded a similar turnover rate to that with Mg2+, but Mn2+ gave a slower rate. Single turnovers of EcoRV on this substrate were analysed by stopped-flow and quench-flow methods, to determine the rates for the formation of the ternary enzyme-DNA-metal complex, the hydrolysis of the phosphodiester bonds and the dissociation of the cleaved DNA. With Co2+, all three steps had similar rates to those with Mg2+. In contrast, Mn2+ gave a faster rate for phosphodiester hydrolysis than either Mg2+ or Co2+, but a slower rate for product dissociation, thus accounting for its low turnover rate. Single turnovers on plasmids also yielded faster rates for substrate hydrolysis with Mn2+ compared to Mg2+ and Co2+. Since Mn2+ gave the most rapid rates for the hydrolytic step, despite being less electronegative than Co2+, the function of the metal ion at the active site of EcoRV cannot be just the polarisation of the scissile phosphate. Moreover, the minimal scheme for the Co2+-catalysed reaction requires two metal ions for DNA cleavage. The metal ions seem to be involved in the precise positioning of both the substrate and the water that acts as the attacking nucleophile and in activating that water molecule. A model is presented to account for how two metal ions might fulfil these functions.
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PMID:DNA cleavage by the EcoRV restriction endonuclease: roles of divalent metal ions in specificity and catalysis. 1032 28

To characterise the pH dependence of phosphodiester hydrolysis by the EcoRV endonuclease in the presence of Mn2+, single turnover reactions on a 12 bp DNA substrate were examined by stopped-flow and quench-flow methods between pH 6.0 and 8.5. At each pH value, the apparent rate constants for phosphodiester hydrolysis increased hyperbolically with the concentration of MnCl2, thus allowing values to be determined for the intrinsic rate constant at saturation with Mn2+ and the equilibrium dissociation constant for Mn2+. The equilibrium constants showed no systematic variation across the pH range tested, while the rate constants increased steeply with increasing pH up to an asymptote above pH 7.5. At low pH conditions, the gradient of a plot of log (rate constant) against pH approached a value of 2. DNA cleavage by EcoRV thus requires the de-protonation of two acidic groups. To determine whether aspartate 36 is one of the groups, mutants of EcoRV were made with other amino acid residues at position 36. Glutamate caused a partial loss of activity, while all other replacements gave near-zero activities. In contrast to wild-type EcoRV, the mutant with glutamate required the de-protonation of only one acidic group for DNA cleavage. A mechanism for EcoRV is proposed in which the water molecule that hydrolyses the phosphodiester bond is de-protonated by two Bronsted bases, probably the ionised forms of aspartate 36 and glutamate 45.
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PMID:DNA cleavage by the EcoRV restriction endonuclease: pH dependence and proton transfers in catalysis. 1032 29

Serratia endonuclease is an important member of a class of magnesium dependent nucleases that are widely distributed in nature. Here, we describe the location and geometry of a magnesium-water cluster within the active site of this enzyme. The sole protein ligand of the magnesium atom is Asn119; this metal ion is also associated with five water molecules to complete an octahedral coordination complex. These water molecules are very well ordered and there is no evidence of rotational disorder or motion. Glu127 and His89 are located nearby and each is hydrogen bonded to water molecules in the coordination sphere. Asp86 is not chelated to the magnesium or its surrounding water molecules. Results of kinetics and site-specific mutagenesis experiments suggest that this metal-water cluster contains the catalytic metal ion of this enzyme. All residues which hydrogen bond to the water molecules that coordinate the magnesium atom are conserved in nucleases homologous to Serratia endonuclease, suggesting that the water cluster is a conserved feature of this family of enzymes. We offer a detailed structural comparison to one other nuclease, the homing endonuclease I-PpoI, that has recently been shown, in spite of a lack of sequence homology, to share a similar active site geometry to Serratia endonuclease. Evidence from both of these structures suggests that the magnesium of Serratia nuclease participates in catalysis via an inner sphere mechanism.
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PMID:The active site of Serratia endonuclease contains a conserved magnesium-water cluster. 1032 93

Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2- at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)N]2-. Dialysis against > or =0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)n]2-. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)n]2- should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.
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PMID:NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9. 1045 17

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


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