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 amino acid residue Asn141 of the restriction endonuclease EcoRI was proposed to make three hydrogen bonds to both adenine residues within the recognition sequence -GAATTC-. We have mutated Asn141 to alanine, aspartate, serine, and tyrosine. Only the serine mutant is active under normal buffer conditions although 1000-fold less than wild-type EcoRI. The alanine and aspartate mutants can be activated by Mn2+. At acidic pH the latter mutant becomes even more active than the wild-type enzyme in the presence of Mn2+. We conclude that Asn141 is essential for DNA recognition and that serine can partly substitute it.
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PMID:Asn141 is essential for DNA recognition by EcoRI restriction endonuclease. 982 60

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV x DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occurring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5' side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.
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PMID:Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site. 985 8

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

We have correlated the structural perturbations caused by DNA mismatches with the enzymatic data of the interaction of the restriction endonuclease EcoRI with DNA. Oligonucleotides d(CGAGAATTCTCA5GAXAATTCT) (X = G, A, T) and d(CGCGAATTYGCGT4CGCXAATTCGCG) (Y = C, X = G, T and Y = A, X = T) containing single mismatches within the EcoRI recognition site were characterized by NMR spectroscopy and by their EcoRI substrate properties. UV melting and gel electrophoresis studies confirm that the oligonucleotides form hairpin structures. The presence of either a CT or a CA mismatch results in markedly lower Tm and van't Hoff enthalpies compared with the fully base paired control. NMR imino proton spectra of these hairpins demonstrate that the perturbation caused by the two mispairs or a noncanonical AT pair is localized and limited to one or two base pairs on either side of the perturbation. The DNA hairpin structures containing single mismatches, and to a lesser extent also sequences with a single noncanonical base pair, are substrates for the restriction endonuclease. In addition to the strand scission at the nonperturbed GpA phosphodiester bond some cleavage is observed at the mismatched position. The interactions of the CA and CT mismatched hairpin with the enzyme are characterized by binding constants that are only 33 and 57 times lower, respectively, than that for the canonical sequence, corresponding to 8-10 kJ x mol(-1) less favorable free binding energy. This, taken together with the NMR data, indicates that the CA and CT mismatches have only small effects on the EcoRI recognition of the DNA substrate. We conclude that two out of the three hydrogen bonds that characterize the interaction of EcoRI with the CG base pair in the canonical sequence can still be formed for either the CT or CA mismatched recognition site.
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PMID:NMR spectroscopic and enzymatic studies of DNA hairpins containing mismatches in the EcoRI recognition site. 992 8

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1(+) [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.
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PMID:The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis. 1002 67

Bovine pancreatic deoxyribonuclease I (DNase I) is an endonuclease which cleaves double-stranded DNA. Cocrystal structures of DNase I with oligonucleotides have revealed interactions between the side chains of several amino acids (N74, R111, N170, S206, T207, and Y211) and the DNA phosphates. The effects these interactions have on enzyme catalysis and DNA hydrolysis selectivity have been investigated by site-directed mutagenesis. Mutations to R111, N170, T207, and Y211 severely compromised activity toward both DNA and a small chromophoric substrate. A hydrogen bond between R111 (which interacts with the phosphate immediately 5' to the cutting site) and the essential amino acid H134 is probably required to maintain this histidine in the correct orientation for efficient hydrolysis. Both T207 and Y211 bind to the phosphate immediately 3' to the cleavage site. Additionally, T207 is involved in binding an essential, structural, calcium ion, and Y211 is the nearest neighbor to D212, a critical catalytic residue. N170 interacts with the scissile phosphate and appears to play a direct role in the catalytic mechanism. The mutation N74D, which interacts with a phosphate twice removed from the scissile group, strongly reduced DNA hydrolysis. However, a comparison of DNase I variants from several species suggests that certain amino acids, which allow interaction with phosphates (positively charged or hydrogen bonding), are tolerated. S206, which binds to a DNA phosphate two positions away from the cleavage site, appears to play a relatively unimportant role. None of the enzyme variants, including a triple mutation in which N74, R111, and Y211 were altered, affected DNA hydrolysis selectivity. This suggests that phosphate binding residues play no role in the selection of DNA substrates.
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PMID:Site-directed mutagenesis of phosphate-contacting amino acids of bovine pancreatic deoxyribonuclease I. 1019 1

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

Apurinic/apyrimidinic endonuclease (APE alias Ref-1) is a multifunctional enzyme involved in DNA repair and redox regulation of transcription factors (e.g., AP-1). It also acts as a repressor of its own and other genes. Recently, it was shown that the level of APE mRNA and protein is enhanced upon treatment of cells with oxidative agents, such as hydrogen peroxide (H(2)O(2)), which gives rise to an adaptive response of cells to oxidative stress. Induction of APE is due to APE promoter activation. To elucidate the mechanism of transcriptional activation of APE by oxidative agents, we introduced mutations into the cloned human APE promoter and checked its activity in transient transfection assays. Here we demonstrate that mutational inactivation of a CREB binding site (CRE) present within the promoter completely abolished APE promoter activation by H(2)O(2), indicating that CREB is required for APE induction. The CRE element in the context of the APE promoter sequence binds c-Jun and ATF-2, which was shown in gel retardation experiments. Under conditions of induction of APE by H(2)O(2), the expression of c-Jun was significantly enhanced, which supports the view that induction of c-Jun is involved in signaling leading to APE promoter activation by oxidative stress.
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PMID:Transcriptional activation of apurinic/apyrimidinic endonuclease (Ape, Ref-1) by oxidative stress requires CREB. 1044 16

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.
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PMID:Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III. 1054 Jul 38

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg2+ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.
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PMID:Application of oligonucleoside methylphosphonates in the studies on phosphodiester hydrolysis by Serratia endonuclease. 1054 47


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