Single-Strand-Specific Nucleases

Gite, Sadanand; Shankar, V.

doi:10.3109/10408419509113536

Cited by 45 publications

(38 citation statements)

References 184 publications

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“…1), it was unclear whether the endonuclease activity of CSP41 was specific for single-stranded or doublestranded RNA. It was also unknown whether CSP41 could digest DNA, as other endonucleases do (21). To test CSP41 specificity, uniformly labeled 121-nt single-and 116-base pair double-stranded RNAs and 5Ј-end-labeled 89-nt single-and 94-base pair double-stranded DNAs were provided as substrates under our standard conditions.…”

Section: Csp41mentioning

confidence: 99%

The Spinach Chloroplast Endoribonuclease CSP41 Cleaves the 3′-Untranslated Region of petD mRNA Primarily within Its Terminal Stem-Loop Structure

Yang

Stern

1997

Journal of Biological Chemistry

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3-Untranslated region stem-loop structures are major determinants of chloroplast mRNA stability. The 3 stem-loop region of spinach petD precursor mRNA (pre-mRNA), a chloroplast gene encoding subunit IV of the cytochrome b 6 ⅐f complex, forms a stable RNA-protein complex in vitro with chloroplast stem-loop binding proteins (CSPs) of 55, 41, and 29 kDa. We have previously purified CSP41 and cloned the corresponding cDNA. In vitro studies demonstrated that CSP41 is a bifunctional protein that displays both endoribonuclease and RNAbinding activities. In this work, the RNase activity of CSP41 is further characterized using the bacterially expressed protein. Our data show that CSP41 cleaves both single-stranded and double-stranded RNAs but not DNA. However, it exhibits a preference for stem-loopcontaining RNAs. When the 3-untranslated region of petD pre-mRNA is provided as a substrate, CSP41 specifically cleaves it within the stem-loop region, implying that CSP41 has an important role in the control of petD mRNA stability. Our data also show that the sequencespecific RNA-binding activity of CSP41 affects the rate, but not the specificity, of its RNase activity, suggesting that CSP41 is probably involved in other events of chloroplast RNA metabolism in addition to RNA degradation. By analyzing C-terminal deletions of CSP41, the RNase domain was located between amino acid residues 73 and 191.Most chloroplast precursor mRNAs (pre-mRNAs) and mature mRNAs contain an inverted repeat sequence in their 3Ј-untranslated region (UTR) 1 that can fold into a stable stemloop structure. Both in vitro and in vivo studies have demonstrated that these 3Ј stem-loop structures are required for correct 3Ј processing of pre-mRNAs and for stabilization of mature mRNAs, by impeding processive 3Ј to 5Ј exonucleolytic degradation. In the absence of a stable secondary structure, or by readthrough of the stem-loop structure, mRNA becomes heterogeneous and generally unstable (1-7). Therefore, the 3Ј stem-loop structure has a primary role in controlling chloroplast mRNA accumulation through pre-mRNA processing and the protection of upstream sequences. Removal of the 3Ј stemloop structure, therefore, is likely to be a key regulatory step in chloroplast mRNA degradation.Bacterial pre-and mature mRNAs are another major group of mRNAs containing 3Ј-inverted repeats. The 3Ј stem-loop structures of bacterial mRNAs function in the processing of pre-mRNAs and the stabilization of mature mRNAs by a mechanism similar to that found in chloroplasts (8). Extensive studies have shown that at least three groups of proteins are involved in the control of 3Ј to 5Ј degradation of bacterial mRNAs: 1) endoribonucleases, such as RNase E and RNase III, which cleave the 3Ј-UTR within or upstream of the stem-loop structure to overcome the secondary structure barrier; 2) exoribonucleases, such as RNase II and polynucleotide phosphorylase, which bind to the free 3Ј end created by the endoribonucleases and degrade mRNA in the 3Ј to 5Ј direction; and 3) stem-loopbinding pr...

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Section: Csp41mentioning

confidence: 99%

The Spinach Chloroplast Endoribonuclease CSP41 Cleaves the 3′-Untranslated Region of petD mRNA Primarily within Its Terminal Stem-Loop Structure

Yang

Stern

1997

Journal of Biological Chemistry

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“…Their application depends on the specificity and mode of action of a particular enzyme. Nucleases have been employed in the production of flavor enhancers, removal of nucleic acids, as therapeutic agents, and for the determination of nucleic acid structure, in particular, mutation typing (Reddy and Shankar 1993;Gite and Shankar 1995;Jasin 1996;Singwi and Joshi 2000;Williams 2001). Nucleases with novel and unusual properties may facilitate the development of advanced technologies in many areas of biotechnology and biomedicine.…”

mentioning

confidence: 99%

A Novel Method for SNP Detection Using a New Duplex-Specific Nuclease From Crab Hepatopancreas

Shagin¹,

Rebrikov²,

Kozhemyako³

et al. 2002

Genome Res.

229

198

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We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed "duplex-specific nuclease preference" (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA.

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“…Thus, it is highly likely that BFN1 is modified post-translationally by the addition of two or three carbohydrate moieties. A number of the other nucleases, including nuclease S1 (Iwamatsu et al, 1991) and nucleases from mung bean (Laskowski, 1980), pea seed (Naseem et al, 1987), barley seed Ho, 1986, 1987), rye germ ribosome (Siwecka et al, 1989), and spinach (Strickland et al, 1991), are glycoproteins, with carbohydrate contents that account for 17% to 29% of the final relative M r (for review, see Gite and Shankar, 1995). Now that the BFN1 gene has been isolated and its activity identified, the role of its potential glycosylation sites and its location within the secretory system can be investigated.…”

mentioning

confidence: 99%

“…All living organisms contain enzymes responsible for the degradation of single-stranded nucleic acids (Gite and Shankar, 1995), including nuclease I proteins (EC 3.1.30.1). Nuclease I enzymes are extracellular heat-stable glycoproteins that degrade both RNA and single-stranded DNA endonucleolytically.…”

mentioning

confidence: 99%

Identification of BFN1, a Bifunctional Nuclease Induced during Leaf and Stem Senescence in Arabidopsis

et al. 2000

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Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

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Single-Strand-Specific Nucleases

Cited by 45 publications

References 184 publications

The Spinach Chloroplast Endoribonuclease CSP41 Cleaves the 3′-Untranslated Region of petD mRNA Primarily within Its Terminal Stem-Loop Structure

The Spinach Chloroplast Endoribonuclease CSP41 Cleaves the 3′-Untranslated Region of petD mRNA Primarily within Its Terminal Stem-Loop Structure

A Novel Method for SNP Detection Using a New Duplex-Specific Nuclease From Crab Hepatopancreas

Identification of BFN1, a Bifunctional Nuclease Induced during Leaf and Stem Senescence in Arabidopsis

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