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)

A physical map of the genome of Rhizobium meliloti 1021 is presented. The physical sizes of the three replicons in this genome had previously been determined and are as follows: the chromosome, 3.4 Mb; pSym-b, 1.7 Mb; and pSym-a, 1.4 Mb. The physical maps for this GC-rich genome contain AT-rich restriction sites for SwaI (5'-TAAATTTA-3'), PacI (5'-TTAATTAA-3'), PmeI (5'-GTTTAAAC-3'), and, for pSym-b, SpeI (5'-ACTAGT-3'). In addition, the endonuclease I-CeuI cleaved the 23S rRNA genes in this genome, and perhaps in most eubacterial genomes. I-CeuI digestion and polymerase chain reaction amplification of rrn regions were used to determine that there are at least three rrn loci in R. meliloti, all of which are located on the chromosome. The orientation of the rrn loci was determined by Southern blotting with probes from rrn sequences located 5' and 3' to the I-CeuI site. The rrn loci are clustered in one part of the chromosome and are oriented so that transcription will occur away from a single point in the circle, as observed for the origin of replication in the Escherichia coli and Salmonella typhimurium chromosomes. Fifteen genes that had been tagged by Tn5 insertion were localized to fragments on the chromosome physical map by using the IS50 as a probe in Southern blots. In addition, glt and gap were placed on the physical map by using Southern hybridization with cloned genes. The fortuitous occurrence of SpecI site in Tn5-233 was used to physically map 10 genetically mapped Tn5-233 integrations on pSym-b and to anchor the physical map to the genetic map. Finally, we demonstrate the usefulness of the map by localizing a total of 12 previously unmapped transposon insertions in the genome. This is the first physical map of the genome of a multireplicon member of the family Rhizobiaceae as well as the first physical map of a Rhizobium chromosome.
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PMID:Physical map of the genome of Rhizobium meliloti 1021. 822 38

Control of Helminthosporium solani, the cause of silver scurf in potato tubers, has been impaired by selection of benzimidazole-resistant strains as a result of repeated use of the fungicide thiabendazole. Identification of thiabendazole-resistant strains of H. solani by conventional techniques takes several weeks. Primers designed from conserved regions of the fungal beta-tubulin gene were used to PCR amplify and sequence a portion of the gene. A point mutation was detected at codon 198 in thiabendazole-resistant isolates causing a change in the amino acid sequence from glutamic acid to alanine or glutamine. Species-specific PCR primers designed to amplify this region were used in conjunction with a restriction endonuclease to cause cleavage in sensitive isolates only and thus provide a rapid diagnostic test to differentiate field isolates.
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PMID:A PCR-based method to characterise and identify benzimidazole resistance in Helminthosporium solani. 923 30

Familial hypercholesterolaemia (FH) is an inherited autosomal codominant disorder caused by many different mutations in the low-density lipoprotein receptor (LDLR) gene. The one described most frequently in patients with FH from England, arises from a G-->A transition at the first nucleotide of codon 80, resulting in the substitution of lysine for glutamic acid at residue 80 of the mature protein, FH E80K. We describe a simple method to detect this mutation in genomic DNA using the polymerase chain reaction (PCR). A 69 base pair (bp) fragment of exon 3 of the LDLR gene is amplified using a mutagenic upstream PCR primer. This substitutes a T for an A residue in the amplified product, 2 bp upstream from the mutant site, generating a restriction site for the endonuclease Taq I, in normal, but not in mutant DNA. Following digestion of amplified DNA with Taq I, normal but not mutant DNA is cut into two fragments of 29 and 40 bp, which are readily identified by polyacrylamide gel electrophoresis. Using this method, 410 patients with clinically diagnosed FH, attending lipid clinics in Edinburgh (72), Newport (158), Walsall (30) and Southampton (150), were screened for the mutation. Five individuals tested positive as heterozygotes, one from Edinburgh, three from Newport and one from Southampton. This finding was confirmed by DNA sequence analysis. We conclude that FH due to this mutation occurs in individuals throughout Great Britain and that it can be detected accurately using this simple technique. DNA from these and other individuals previously identified to be heterozygous for FH E80K, was then studied using PCR of highly informative microsatellite markers flanking the LDLR gene. Sixteen of 17 apparently unrelated individuals heterozygous for FH E80K also were heterozygous for an identical size (239 nucleotide) allele, of polymorphic microsatellite D19S394, located approximately 250 kb away from the LDLR gene. This supports the hypothesis that FH E80K in these 16 individuals arose from a single ancestor less than 1000 years ago.
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PMID:Simplified detection of a mutation causing familial hypercholesterolaemia throughout Britain: evidence for an origin in a common distant ancestor. 954 93

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

Terminase, an enzyme encoded by the Nu1 and A genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions. cosN, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although cosN is rotationally symmetric, mutations in cosN have asymmetric effects. The cosN G2C mutation (a G-to-C change at position 2) in the left half of cosN reduces the phage yield 10-fold, whereas the symmetric mutation cosN C11G, in the right half of cosN, does not affect the burst size. The reduction in phage yield caused by cosN G2C is correlated with a defect in cos cleavage. Three suppressors of the cosN G2C mutation, A-E515G, A-N509K, and A-R504C, have been isolated that restore the yield of lambda cosN G2C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. lambda A-E515G, A-N509K, and A-R504C phages, which are cosN+, also had wild-type burst sizes. In vitro cos cleavage experiments on cosN G2C C11G DNA showed that the rate of cleavage for A-E515G terminase is three- to fourfold higher than for wild-type terminase. The A-E515G mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of lambda cosN G2C C11G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of lambda cosN G2C C11G. In a lambda cosN+ background, all amino acids tested at position 515 were functional. These results suggest that A-E515G plays an indirect role in extending the specificity of the endonuclease activity of lambda terminase.
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PMID:Mutations that extend the specificity of the endonuclease activity of lambda terminase. 986 33

Target sequence-specific DNA binding regions of the restriction endonuclease EcoRII were identified by screening a membrane-bound EcoRII-derived peptide scan with an EcoRII recognition site (CCWGG) oligonucleotide duplex. Dodecapeptides overlapping by nine amino acids and representing the complete protein were prepared by spot synthesis. Two separate DNA binding regions, amino acids 88-102 and amino acids 256-273, which share the consensus motif KXRXXK, emerged. Screening 570 single substitution analogues obtained by exchanging every residue of both binding sites for all other amino acids demonstrated that replacing basic residues in the consensus motifs significantly reduced DNA binding. EcoRII mutant enzymes generated by substituting alanine or glutamic acid for the consensus lysine residues in DNA binding site I expressed attenuated DNA binding, whereas corresponding substitutions in DNA binding site II caused impaired cleavage, but enzyme secondary structure was unaffected. Furthermore, Glu96, which is part of a potential catalytic motif and also locates to DNA binding site I, was demonstrated to be critical for DNA cleavage and binding. Homology studies of DNA binding site II revealed strong local homology to SsoII (recognition sequence, CCNGG) and patterns of sequence conservation, suggesting the existence of functionally related DNA binding sites in diverse restriction endonucleases with recognition sequences containing terminal C:G or G:C pairs.
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PMID:Regions of endonuclease EcoRII involved in DNA target recognition identified by membrane-bound peptide repertoires. 998 71

During the course of work aimed at isolating a rice gene from Oryza australiensis by PCR, the oligonucleotide primers used were found to generate a fragment that showed sequence homology to the endonuclease (EN) region of the maize non-LTR retrotransposon (LINE) Cin4. We carried out further PCRs using oligonucleotide primers that hybridized to these sequences, and found that they amplified several fragments, each with homology to the EN regions, from Oryza sativa cv. Nipponbare as well as O. australiensis. We mapped the approximate locations of two rice LINE homologues by screening clones in a YAC library made from a rice (O. sativa) genome, and found that each homologue was present in a low copy number apparently at nonspecific regions on rice chromosomes. We then carried out PCR using degenerate oligonucleotide primers which hybridized to the rice LINE homologues and Cin4 to ascertain whether LINE homologues are present in a variety of members of the plant kingdom, including angiosperms, gymnosperms, bracken, horsetail and liverwort. Cloning and nucleotide sequencing revealed that 53 clones obtained from 27 out of 33 plant species contained LINE homologues. In addition to these homologues, we identified four homologues with EN regions in the Arabidopsis thaliana genome by a computer search of databases. The nucleotide sequences of almost all the LINE homologues were greatly diverged, but the derived amino acid sequences were well conserved, and all contained glutamic acid and tyrosine residues at almost the same relative positions as in the the active site regions of AP (apurinic/apyrimidinic)-endonucleases. The EN regions in the LINE homologues from closely related plant species show a closer phylogenetic relationship, indicating that sequence divergence during vertical transmission has been a major influence upon the evolution of plant LINEs.
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PMID:Non-LTR retrotransposons (LINEs) as ubiquitous components of plant genomes. 1007 Dec 12

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

Mutations in DNA mismatch repair (MMR) lead to increased mutation rates and higher recombination between similar, but not identical sequences, as well as resistance to certain DNA methylating agents. Recently, a component of human MMR machinery, MutLalpha, has been shown to display a latent endonuclease activity. The endonuclease active site appears to include a conserved motif, DQHA(X)(2)E(X)(4)E, within the COOH-terminus of human PMS2. Substitution of the glutamic acid residue (E705) abolished the endonuclease activity and mismatch-dependent excision in vitro. Previously, we showed that the PMS2-E705K mutation and the corresponding mutation in Saccharomyces cerevisiae were both recessive loss of function alleles for mutation avoidance in vivo. Here, we show that mutations impacting this endonuclease motif also significantly affect MMR-dependent suppression of homeologous recombination in yeast and responses to S(n)1-type methylating agents in both yeast and mammalian cells. Thus, our in vivo results suggest that the endonuclease activity of MutLalpha is important not only in MMR-dependent mutation avoidance but also for recombination and damage response functions.
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PMID:Mutations affecting a putative MutLalpha endonuclease motif impact multiple mismatch repair functions. 1756 44

Among the four known mechanisms of intron removal, three are reputedly catalyzed by RNA molecules. In the fourth mechanism, a protein endonuclease removes introns from nuclear tRNA and all archaeal RNAs. Three strictly conserved residues of the splicing endonuclease, a histidine, a lysine, and a tyrosine, were predicted to catalyze the intron cleavage reaction in a manner similar to that of the catalytic triad of ribonuclease A. Single-turnover kinetic parameters were obtained for the wild-type enzyme and two triad mutants. Mutation of histidine to alanine produced an at least approximately 28-fold reduction; mutation of tyrosine to phenylalanine produced an at least approximately 7-fold reduction in activity, while a histidine and tyrosine double mutation abolished cleavage. The single mutation of lysine to glutamic acid abolished RNA cleavage activity in the absence of a divalent metal but maintained a substantial level of activity in the presence of specific divalent metals. These data support important functional roles already proposed for the catalytic triad and suggest an intriguing hypothesis in which the splicing endonuclease is an intermediate in the transition from the RNA to the RNP world.
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PMID:Probing the catalytic triad of an archaeal RNA splicing endonuclease. 1905 88


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