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At least 1,117 records · Page 62Linked to original sources
PubMed1988

Sequence and expression of NUC1, the gene encoding the mitochondrial nuclease in Saccharomyces cerevisiae.

The DNA sequence and studies on the expression of the NUC1 gene from Saccharomyces cerevisiae are presented. The NUC1 locus is located in the distal portion of the left arm of Chromosome X and encodes the major nuclease found in mitochondria. The inferred amino acid sequence of NUC1 predicts that the nuclease is basic, rich in prolines, of average hydrophobicity, and has a molecular weight for the primary translation product of 37,209 daltons. NUC1 is very poorly expressed, consistent with the codon usage bias determined from the DNA sequence and our previous determination of the number of enzyme molecules per cell. Mapping of the 5' terminus of the NUC1 mRNA reveals that the mRNA has a long 400 base untranslated leader in which are found three open reading frames, each initiated by an AUG. The possibility that these upstream open reading frames contribute to the poor expression of the NUC1 gene is discussed.

Amino Acid Sequence↗
PubMed1989

Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases.

2'-O-Methyloligoribonucleotides have been synthesised on solid phase from base protected 5'-O-dimethoxytrityl-2'-O-methylribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) using 5-(4-nitrophenyl)-1H-tetrazole as activator. Coupling yields greater than 99% were achieved, as judged by trityl cation release. The preparation of a modified 2'-deoxycytidine building block bearing an N4-(5-trifluoroacetylaminopentyl) spacer is also described. The latter compound enabled the chemical synthesis of 2'-O-methyloligoribonucleotide probes carrying several 5'- terminal biotinylation sites (in general four modified residues were used), which can be conveniently 32P end-labelled enzymatically using polynucleotide kinase. Used in conjunction with streptavidin-containing derivatives, such biotinylated probes have important applications in biochemical purification and electron microscopy of RNA-protein complexes. The 2'-O-methyloligoribonucleotides are completely resistant to degradation by either RNA or DNA specific nucleases. In contrast, nucleases with dual RNA/DNA specificity show a complete spectrum of cleavage rates.

Biotin↗
PubMed1990

Structural investigation of the in vitro transcript of the yeast tRNA(phe) precursor by NMR and nuclease mapping.

Both NMR and nuclease mapping have been used to probe the structure of an unmodified yeast tRNA(phe) precursor synthesized in vitro by T7 RNA polymerase. A comparison of the NMR data of the precursor and of the mature tRNA transcript shows that the mature tRNA domain structure is similar in both molecules. In the tRNA precursor, the intron consists of a stem of at least four base-pairs, identified by NMR, and two single-stranded loops, identified by nuclease mapping. This is in agreement with the structure previously proposed for the native tRNA(phe) precursor (1). However, our data also show the intron structure to be less stable than the mature tRNA domain, suggesting that the precursor may best be described as having two domains with a hinge at the junction of the anticodon and intron stems.

Anticodon↗
PubMed1991

Mung bean nuclease cleavage pattern at a polypurine.polypyrimidine sequence upstream from the mouse metallothionein-I gene.

Mung bean nuclease, an enzyme specific for single-stranded DNA, was used to probe a non-B DNA structure present in the mouse metallothionein-I gene. The region sensitive to the enzyme was constituted by a 128 base-pair long polypurine.polypyrimidine sequence located at 1.2-kb from the start of transcription. A detailed analysis of the mung bean nuclease cleavage pattern revealed that: (i) under conditions of supercoiling and low pH a triplex structure was formed, (ii) the triplex was flanked by a sequence with the potential of forming a Z-DNA structure, (iii) most of the enzymatic activity was localized at some of the junctions between double-stranded and triple-stranded DNA and at mismatches in the triplex, (iv) no unpaired bases were observed in the loop or outside the triplex, and (v) the triplex was present in more than one configuration.

Animals↗
PubMed1993

Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.

Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure--function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides at noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (kcat = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (kcat) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.

Animals↗
PubMed1993

4'-Thio-oligo-beta-D-ribonucleotides: synthesis of beta-4'-thio-oligouridylates, nuclease resistance, base pairing properties, and interaction with HIV-1 reverse transcriptase.

We present the synthesis and the study of properties of a new series of modified oligonucleotides, namely 4'-thio-oligo-beta-D-ribonucleotides (4'-S-RNA). Homo-oligonucleotides of this class (4'-SU6 and 4'-SU12) were prepared from the previously known thionucleosides using the phosphoramidite methodology. The comparison of the substrate properties of 4'-SU6 and its natural analog U6 with respect to four nucleases indicates that the former is much more resistant than the latter. Such resistance to nucleases in addition to relatively high Tm values for 4'-SU12 hybridized with Poly(A) show that these new 4'-S-RNA are good candidates for potential antisense effects. The oligonucleotides 4'-SU6 and 4'-SU12 have been also evaluated as non sequence specific inhibitors of HIV-1 reverse transcriptase. All available evidences, based primarily on fluorescence measurements, are consistent with the binding of 4'-SU6 and 4'-SU12 to RT at a site which is different from the polymerase site of the enzyme.

Animals↗
PubMed1993

Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease.

Base Sequence↗
PubMed1996

Detection of HIV-1 DNA in cells and tissue by fluorescent in situ 5'-nuclease assay (FISNA).

The critical aspects of successful in situ amplification include fixation, permeabilization, amplification and detection. We address these aspects and present a novel detection scheme that eliminates hybridization following amplification. We use the 5'-nuclease activity of Taq polymerase to cleave in situ a 5'-reporter dye from an oligonucleotide probe which hybridizes to the target amplicon during amplification. The 5'-reporter dye is disassociated from the 3'-quenching dye and remains localized by charge interactions. In addition, we describe probe design constraints for 5'-nuclease assays both in solution and in situ. Using this technique, we show the sensitive and specific detection of HIV-1 DNA in cells lines and tissue from HIV-1-infected individuals.

5'-Nucleotidase↗
PubMed1996

Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity.

The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.

DNA↗
PubMed1997

Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties.

We report here the site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides (ODNs). ODNs containing both 5-( N-aminohexyl)-carbamoyl-2'-deoxyuridine (H), which serves as a tether for the further conjugation of functional groups, and 5-(N,N-dimethylaminohexyl)carbamoyl-2'-deoxyuridine (D), which contributes to the thermal stability of the duplex and to the resistance to nucleolytic hydrolysis by nucleases, were synthesized. Functional groups such as folic acid and palmitic acid were site-specifically introduced into the terminus of the aminohexyl-linker of H. The thermal stability and resistance toward nuclease digestion of the modified ODNs were studied. We found that ODNs containing D and H formed stable duplexes with both the complementary DNA and RNA strands even when a bulky functional group such as folic acid, palmitic acid or cholesterol was attached to the terminus of the amino-linker. We also found that ODN analogues which contained D were more resistant to nucleolytic degradation by exo- and endonuclease than the unmodified ODN. Furthermore, duplexes formed by ODNs containing D and the complementary RNA could elicit RNase H activity.

Animals↗
PubMed1997

Influence of the nature of the porphyrin ligand on the nuclease activity of metalloporphyrin-oligonucleotide conjugates designed with cationic, hydrophobic or anionic metalloporphyrins.

The synthesis of metalloporphyrin-oligonucleotide conjugates with different metalloporphyrin moieties are described as well as the comparison of their in vitro nuclease efficiency toward a single-stranded DNA target. Between cationic, anionic and hydrophobic manganese porphyrins covalently linked to the oligonucleotide, the best nuclease activity was obtained with the cationic ones, suggesting that the affinity of the cleaver to the DNA target is a key factor.

Anions↗
PubMed1998

Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins.

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of approximately 100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact beta/alpha fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by site-directed mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg2+that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.

Amino Acid Sequence↗
PubMed1999

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications.

Base Sequence↗
PubMed2000

The nature of the 5'-terminus is a major determinant for DNA processing by Schizosaccharomyces pombe Rad2p, a FEN-1 family nuclease.

The nuclease activity of FEN-1 is essential for both DNA replication and repair. Intermediate DNA products formed during these processes possess a variety of structures and termini. We have previously demonstrated that the 5'-->3' exonuclease activity of the Schizosaccharomyces pombe FEN-1 protein Rad2p requires a 5'-phosphoryl moiety to efficiently degrade a nick-containing substrate in a reconstituted alternative excision repair system. Here we report the effect of different 5'-terminal moieties of a variety of DNA substrates on Rad2p activity. We also show that Rad2p possesses a 5'-->3' single-stranded exonuclease activity, similar to Saccharomyces cerevisiae Rad27p and phage T5 5'-->3' exonuclease (also a FEN-1 homolog). FEN-1 nucleases have been associated with the base excision repair pathway, specifically processing cleaved abasic sites. Because several enzymes cleave abasic sites through different mechanisms resulting in different 5'-termini, we investigated the ability of Rad2p to process several different types of cleaved abasic sites. With varying efficiency, Rad2p degrades the products of an abasic site cleaved by Escherichia coli endonuclease III and endonuclease IV (prototype AP endonucleases) and S.POMBE: Uve1p. These results provide important insights into the roles of Rad2p in DNA repair processes in S.POMBE:

Carbon-Oxygen Lyases↗
PubMed2000

Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease.

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N:-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF-, AAF- and AP-DNA adducts, determined by gel mobility shift assay, are 33 +/- 9, 8 +/- 2 and 23 +/- 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF-DNA was cleaved approximately 2-fold more efficiently than AF- or AP-DNA (AAF > AF approximately AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF-DNA (but not for AP-DNA), making it equivalent to that for AAF-DNA. These results are consistent with a model in which DNA damage recognition by the E. coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF- and AAF-DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.

2-Acetylaminofluorene↗
PubMed1977

Prepartation of soluble chromatin and specific chromatin fractions with restriction nucleases.

By digestion of rat liver nuclei with EndoR HaeIII, EndoR EcoRI, and EndoR Bam and subsequent lysis of the nuclei approx. 90%, 40%, and 45%, respectively, of the chromatin were solubilized. The plateau values of solubilization are in agreement with a model in which the chromatin strands are crosslinked and/or attached to a supporting structure. The distribution of DNA lengths in the soluble and insoluble chromatin fractions were determined. According to digestion experiments with restriction nucleases rat liver DNA contains highly repetitive sequences, some of which are arranged in tandem repeats of 95 and 380 nucleotide pairs, respectively. With EndoR EcoRI chromatin containing the repetitive RNA was preferentially solubilized and, by subsequent sucrose gradient centrifugation, purified to about 90%. The useful properties of chromatin prepared by the specific action of restriction nucleases are discussed.

Animals↗
PubMed1977

The specificity of S1 nuclease toward RNA-DNA hybrids as studied using isotopes of phosphorus-32 and phosphorus-33.

Hybrids were formed from Bacillus cereus DNA and ribosomal RNA. They were treated with various combination of S1 nuclease and ribonuclease, and the molar ratios of the RNA and DNA moieties remaining in the treated hybrids were determined using a 32P-33P dual-label technique. It was found that both S1 nuclease and ribonuclease are required to give hybrid with RNA and DNA in a perfect 1:1 molar ratio. It was noted that the dual-label technique which employs orthophosphate as the sole phosphorus source for both labels gives unambiguous molar ratios and obviates the need to calculate specific activities, make quench corrections, or correct for base content.

Bacillus cereus↗
PubMed1978

Purification of potential 3' processing nucleases using synthetic tRNA precursors.

The synthetic tRNA precursors, tRNA-C-114C]U and tRNA-C-C-A-[14C]C-C, as well as poly (a) and diesterase-treated tRNA, have been used to identify and purify potential 3'processing nucleases. Four activities have been separated by this analysis; and three of them have been characterized. Two of the enzymes, which are well-separated on hydroxylapatite columns, act on poly(A), require K+ and Mg2+ for activity, and have molecular weights of about 90,000. These activities have properties previously ascribed to RNase II. The third enzyme does not act on poly(A), requires Mg2+ for activity, and has a molecular weight of about 60,000. It is identical to RNase D, previously characterized as an exonuclease acting on tRNAs with altered structure. Each of the enzymes can remove nucleotides from the tRNA precursor containing extra nucleotides beyond the 3'terminus, whereas they are relatively inactive with intact tRNA or tRNA-C-U. The greatest specificity was displayed by RNase D. The possibility that RNase D is a 3'processing nuclease is discussed.

Base Sequence↗