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 mutational potency of apurinic/apyrimidinic (AP) sites induced by heat-treatment under acidic conditions has been studied in mammalian cells. Abasic sites were induced on a single-stranded DNA shuttle vector carrying the supF tRNA gene, eliminating, therefore, any ambiguity concerning the damaged strand. This vector was able to replicate both in mammalian cells and in bacteria where the mutations induced in animal cells on the supF tRNA gene were screened by the white/blue beta-galactosidase assay in the presence of isopropyl-1-thio-beta-D-galactopyranoside and 5-bromo-4-chloro-3-indoyl-beta-D-galactoside. All white colonies contained plasmid with a mutation on the target gene which was directly sequenced. Our results show that one AP site was induced/22 min of heating as measured by sensitivity of DNA to alkali denaturation or treatment with the AP-endonuclease activity of the FPG protein (Fapy-DNA glycosylase). Putative AP sites decrease survival of the plasmid with a lethal hit of one AP site/single-stranded molecule. Mutation frequency was increased by a factor of approximately six after 2 h at 70 degrees C. Most of the induced mutations were point mutations not distributed at random and clustered in the gene region which will give rise to the mature tRNA. Mutations were abolished by treatments that eliminated AP sites such as alkali treatment or incubation with the Fapy-DNA glycosylase protein. Under our experimental conditions, when only single mutations were taken into account, the order of base insertion opposite AP sites was G greater than A greater than T greater than C.
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PMID:Mutation spectrum of heat-induced abasic sites on a single-stranded shuttle vector replicated in mammalian cells. 152 92

We show here that yeast tRNA ligase protein is essential in the cell and participates in joining together tRNA half-molecules resulting from excision of the intron by the splicing endonuclease. A haploid yeast strain carrying a chromosomal deletion of the ligase gene is viable only if ligase protein can be supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase gene is repressed, cells eventually die and accumulate endonuclease cut but unligated half-molecules and intervening sequences. Half-molecules that accumulate appear to be fully end-processed. Two temperature-sensitive ligase mutant strains have been isolated; these strains accumulate a similar set of unligated half-molecules at the nonpermissive temperature.
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PMID:Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates. 1989 Oct 53

A survey of exon sequence and structure requirements for splicing was undertaken using labeled pre-tRNA substrates prepared by in vitro transcription of bacterial promoter-yeast tRNA(Tyr) gene fusions. Transcription templates were assembled from oligonucleotide cassettes allowing analysis of 22 derivatives affecting each of the potential secondary and certain tertiary interactions in the pre-tRNA. Effects on both excision of the intervening sequence by yeast endonuclease and joining of exons by ligase were examined. Replacements within the D- and T-stems and anticodon stems revealed that while the primary sequences of these segments were not essential for splicing, formation of base-paired structures was required. Replacements which altered the primary sequence while retaining the secondary structure of the aminoacyl stem allowed efficient excision by endonuclease but reduced joining by ligase. Potentially, the effects of changes within these stems may be indirect through effects on adjacent or overall structure. The presence of either structured or unstructured 5' leader and/or 3' trailer sequences had no effect on either splicing step. Alterations in the conserved Levitt tertiary pair (G15/C48), previously implicated in splicing of pre-tRNA(Phe), did not alter splicing of pre-tRNA(Tyr). A precursor in which the small (type I) extra arm in pre-tRNA(Tyr) was replaced with the large (type II) extra arm sequence from tRNA(Ser) was efficiently spliced. These and previous results suggest that only limited features of exon sequence or structure are recognized by the splicing enzymes.
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PMID:Exon sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae. 154 May 92

Introns have generally been assumed to be passive in the transfer RNA splicing reaction. Experiments have now been done showing that the endonuclease is able to cut a precursor provided that a base in the single-stranded loop of the intron can pair with the base of the 5' exon situated at the position that immediately follows the anticodon stem (position 33 in the yeast tRNA isoacceptor pre-tRNA(Leu)3, position 32 in yeast pre-tRNA(Phe)). The elucidation of the role of the intron reveals that in addition to the conserved bases, there are positions in the mature domain which, although not necessarily occupied by the same base in all pre-tRNA's, nevertheless have a fundamental role in the splicing reaction. These positions are termed cardinal positions.
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PMID:Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease. 154 87

Initial amplification and sequencing of a 366-bp fragment of the cytochrome b gene by a conserved primer pair (MVZ 03 and MVZ 04) revealed a nonfunctional copy of the gene with two deletions (one of which is 17 bp in length and the other of which is 3 bp in length) in Chroeomys jelskii, a South American akodontine rodent. By means of an alternative primer to MVZ 03--namely, MVZ 05--from the region of the tRNA for glutamic acid, a functional copy of cytochrome b was subsequently amplified. Both primer pairs amplify functional sequence when applied to purified mitochondrial DNA (mtDNA). Restriction-endonuclease digestion of purified mtDNA from C. jelskii did not reveal any additional sets of bands that would suggest heteroplasmy in the mitochondrial genome. When probed with both functional and nonfunctional gene fragments, MboI restriction digests revealed the same pattern, providing further evidence that the nonfunctional copy must be located in the nucleus. Observed differences in the mitochondrial and nuclear sequences from two populations are consistent with a faster rate of change in mtDNA than in nuclear DNA.
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PMID:Mitochondrial DNA-like sequence in the nuclear genome of an akodontine rodent. 156 Jul 58

We have isolated an endonuclease from E. coli active on bleomycin-treated DNA. Purification on DEAE-cellulose separated this activity in strains lacking endonuclease I, endonuclease III or exonuclease III. After DEAE chromatography, the enzyme was active in the absence of divalent cations and was not inhibited by tRNA or harmane. In addition, this enzyme was stable at 45 degrees C for 20 min. These properties are consistent with this activity being endonuclease IV. This was supported by our finding no activity in a strain lacking endonuclease IV.
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PMID:Bleomycin-treated DNA is specifically cleaved only by endonuclease IV in E. coli. 168 11

The intron-containing tRNA(Trp) precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs. To investigate the role of this structure in cleavage by the halophilic endonuclease, a series of mutant tRNA(Trp) RNAs were prepared and evaluated as substrates. We find that alterations in this structure result in the loss of cleavage at both 5' and 3' sites. Cleavage of a 35-nucleotide model RNA substrate, containing only these features, demonstrates that sequences and structures present at the exon-intron boundaries are sufficient for recognition and cleavage. We have also examined the mechanism used by the halophilic endonuclease to identify the cleavage sites. Addition of a single base, or a base pair in the anticodon stem above the cleavage sites, does not affect the cleavage site selection. The addition of nucleotides between the two cleavage sites significantly decreases cleavage efficiency and has an effect on the cleavage site selection. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases.
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PMID:Recognition of exon-intron boundaries by the Halobacterium volcanii tRNA intron endonuclease. 169 85

tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.
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PMID:Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases. 173 50

In an Escherichia coli expression system, two genes, one from an anaerobic intestinal bacterium and one from E. coli, were overexpressed following the alteration of ribosome-binding (Shine-Dalgarno) sequences. For both genes, the polymerase chain reaction (PCR) was used to modify the ribosome-binding sequence and, at the same time, provide restriction endonuclease sequences at each end of the gene. These restriction endonuclease sequences were used for inserting the DNA into the E. coli plasmid vector pGEM2, which has the T7 promoter upstream from its multiple cloning sites. Each chimeric plasmid, made by ligating the PCR product into pGEM2, was transformed into E. coli strain HMS174(DE3) which, when induced, produces T7 RNA polymerase for regulated overexpression. The gene isolated from the anaerobic intestinal bacterium, a 27-kDa polypeptide gene from Eubacterium sp. strain 12708, when expressed using this system, produced about one-third of the total cell protein as measured in Coomassie-stained protein gels and confirmed by Western blots with rabbit antibody. The E. coli enzyme, a 28.4-kDa tRNA methylation enzyme, was increased fivefold in activity of cell extracts over that of the best previous strain.
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PMID:Rapid method for altering bacterial ribosome-binding sequences for overexpression of proteins in Escherichia coli. 182 79

Fusion of a prokaryotic promoter to a yeast tRNA gene provides a means for uncoupling analyses of mutations affecting splicing from requirements for transcription and other processing steps. For this purpose, a phage lambda promoter was fused to the Saccharomyces cerevisiae tRNATyr(SUP3a) coding sequence. This fusion allows the synthesis of an end-mature precursor by in vitro transcription with Escherichia coli RNA polymerase. This precursor was accurately spliced by purified yeast endonuclease and ligase fractions. However, both the initial rate and the extent of the endonuclease cleavage reaction were reduced in comparison to those for substrates produced by yeast RNA polymerase III. Efficient splicing could be restored in a magnesium- and temperature-dependent renaturation step, suggesting a conformational transition was required. Enzymatic solution structure probing of transcripts from wild-type and intron-variant templates revealed that the essential conformational transition involved a segment of the tRNA-like portion of the precursor. These results (1) suggest that the primary sequence of the precursor alone may not be sufficient to ensure formation of the active conformer during synthesis, (2) provide direct evidence that endonuclease recognizes mature tRNA-like structure in the precursor, and (3) suggest a general caution for the use of semisynthetic transcripts in RNA processing reactions. Potentially, transcription and processing of tRNATyr in yeast may provide a useful paradigm for examining active control of conformation in RNA biosynthesis.
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PMID:Conformational transition required for efficient splicing of transcripts from hybrid lambda promoter yeast tRNA gene fusion. 182 90


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