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 Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.
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PMID:Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI). 894 51

A genomic bank from Brevibacterium divaricatum has been prepared using lambda EMBL3 as a vector. The genomic bank's titers are 2.2 x 10(6) pfu/micrograms. Through screening by plaque hybridization, a 9.6 kb NcoI fragment which contains the entire trp operon has been isolated. Polymerase chain reaction amplification and restriction endonuclease analysis of PCR fragments indicated that there is homology between the coryneform bacteria; however, some genetic diversity among the species still exists. By complementation tests using subcloning of the 9.6 kb NcoI fragments and various E. coli tryptophan auxotrophs, this fragment was found to contain a gene cluster composed of trpE, trpD, trpC, trpB and trpA in this order. This revealed that the tryptophan biosynthesis genes in B. divaricatum may be an operon.
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PMID:Cloning of the tryptophan operon of Brevibacterium divaricatum and its expression in E. coli. 895 24

Acute intermittent porphyria (AIP) is an inherited disorder characterized by a deficiency of hydroxymethylbilane synthase (EC 4.3.1.8.; HMBS), the third enzyme in the heme biosynthetic pathway. To date, 113 different HMBS gene mutations have been reported in the world. However, there were a few reports of the gene mutations in the Japanese AIP patients. We studied the gene mutation in two unrelated AIP families in the San-in district, a local area of Western Japan. The overlapping 6 fragments of the HMBS gene, amplified by the reverse transcript-polymerase chain reaction, were analyzed by the single-strand conformation polymorphism with silver staining technique. The abnormal fragment from a member of one family was sequenced to detect the C to T substitution at 517 nucleic acid position of cDNA, which led to a missense mutation of arginine to tryptophan exchange at an amino acid level (R173W). This mutation located in exon 10 created a new site of the MSP 1 restriction endonuclease and was screened by the amplified fragment of exon 10 from genomic DNA with the MSP 1 digestion. The mutation was detected totally in three members of the family and interestingly also in two patients of an unrelated family. This mutation has been reported widely in the world independently, such as in a Swedish, a Canadian, a Finnish, and a French family, but is the first in Japanese patients. The screening method for this mutation is useful for diagnosis in Japanese AIP patients.
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PMID:Mutation in the exon 10 (R173W) of the hydroxymethylbilane synthase gene in two unrelated Japanese families with acute intermittent porphyria. 952 50

The circular, 17,443 nucleotide-pair mitochondrial (mt) DNA molecule of the sea anemone, Metridium senile (class Anthozoa, phylum Cnidaria) is presented. This molecule contains genes for 13 energy pathway proteins and two ribosomal (r) RNAs but, relative to other metazoan mtDNAs, has two unique features: only two transfer RNAs (tRNA(f-Met) and tRNA(Trp)) are encoded, and the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes each include a group I intron. The COI intron encodes a putative homing endonuclease, and the ND5 intron contains the molecule's ND1 and ND3 genes. Most of the unusual characteristics of other metazoan mtDNAs are not found in M. senile mtDNA: unorthodox translation initiation codons and partial translation termination codons are absent, the use of TGA to specify tryptophan is the only genetic code modification, and both encoded tRNAs have primary and secondary structures closely resembling those of standard tRNAs. Also, with regard to size and secondary structure potential, the mt-s-rRNA and mt-1-rRNA have the least deviation from Escherichia coli 16S and 23S rRNAs of all known metazoan mt-rRNAs. These observations indicate that most of the genetic variations previously reported in metazoan mtDNAs developed after Cnidaria diverged from the common ancestral line of all other Metazoa.
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PMID:The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns, a paucity of tRNA genes, and a near-standard genetic code. 953 27

Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase.
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PMID:Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis. 966 81

The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure.
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PMID:Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease. 1007 6

Structural studies of the proteins of the BstVI restriction-modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 degrees C and 55 or 60 degrees C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3-5 microM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 microM at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 degrees C and 55 degrees C. These results were interpreted in terms of differences in the structural characteristics of these restriction-modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 degrees C and at the temperature at which they are most active.
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PMID:Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy. 1042 88

Gaucher disease is the most prevalent inherited sphingolipidosis and results from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1, or non-neuronopathic; type 2, or acute neuronopathic; and type 3, or subacute neuronopathic. We have identified a novel mutation in a patient of Russian-British descent who died of type 2 Gaucher disease a few hours after birth. A heterozygous T --> C transition mutation in exon 6, cDNA nucleotide position 667, results in the substitution of tryptophan by arginine at amino acid residue 184 (W184R) of glucocerebrosidase. This mutation creates a new cleavage site for the restriction endonuclease Hinf1. We developed a method that utilizes Hinf1 restriction endonuclease analysis to confirm the presence of the mutation and test family members. The second mutation identified in the other glucocerebrosidase allele of the patient is mutation L444P, a severe mutation frequent in type 2 and 3 Gaucher disease. Since the patient died very shortly after birth, we postulate that the W184R/L444P genotype may result in little or no detectable glucocerebrosidase activity and thus a poor prognosis.
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PMID:Novel point mutation (W184R) in neonatal type 2 Gaucher disease. 1067 38

EcoRI endonuclease has two tryptophans at positions 104 and 246 on the protein surface. A single tryptophan mutant containing Trp246 and a single cysteine labeling site at the N-terminus was used to determine the position of the N-terminus in the protein structure. The N-termini of EcoRI endonuclease are essential for tight binding and catalysis yet are not resolved in any of the crystal structures. Resonance energy transfer was used to measure the distance from Trp246 donor to IAEDANS or MIANS acceptors at Cys3. The distance is 36 A in apoenzyme, decreasing to 26 A in the DNA complex. Molecular modeling suggests that the N-termini are located at the dimer interface formed by the loops comprising residues 221-232. Protein conformational changes upon binding of cognate DNA and cofactor Mg(2+) were monitored by tryptophan fluorescence of the single tryptophan mutant and wild-type endonuclease. The fluorescence decay of Trp246 is a triple exponential with lifetimes of 7, 3.5, and 0.7 ns. The decay-associated spectra of the 7- and 3.5-ns components have emission maxima at approximately 345 and approximately 338 nm in apoenzyme, which shift to approximately 340 and approximately 348 nm in the DNA complex. The fluorescence quantum yield of the single tryptophan mutant drops 30% in the DNA complex, as compared to 10% for wild-type endonuclease. Fluorescence changes of Trp104 upon binding of DNA were inferred by comparison of the decay-associated spectra of wild type and single tryptophan mutant. Fluorescence changes are related to changes in proximity and orientation of quenching functional groups in the tryptophan microenvironments, as seen in the crystal structures.
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PMID:Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor. 1117 Mar 85

The cap-dependent endonuclease of the influenza viral RNA polymerase, which produces the capped RNA primers that initiate viral mRNA synthesis, is comprised of two active sites, one for cap binding and one for endonuclease cleavage. We identify the amino acid sequences that constitute these two active sites and demonstrate that they are located on different polymerase subunits. Binding of the 5' terminal sequence of virion RNA (vRNA) to the polymerase activates a tryptophan-rich, cap-binding sequence on the PB2 subunit. At least one of the tryptophans functions in cap binding, indicating that this active site is probably similar to that of other known cap-binding proteins. Endonuclease cleavage, which is activated by the subsequent binding of the 3' terminal sequence of vRNA, resides in a PB1 sequence that contains three essential acidic amino acids, similar to the active sites of other enzymes that cut polynucleotides to produce 3'-OH ends. These results, coupled with those of our previous study, provide a molecular map of the five known essential active sites of the influenza viral polymerase.
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PMID:The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. 1129 40


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