MersV1, Istex, Corpus, bibRecord, 000823

Site-Specific Ribonuclease Activity of Eukaryotic DNA Topoisomerase I

Identifieur interne : 000823 ( Istex/Corpus ); précédent : 000822; suivant : 000824

Site-Specific Ribonuclease Activity of Eukaryotic DNA Topoisomerase I

Auteurs : Joann Sekiguchi ; Stewart Shuman

Source :

Molecular Cell [ 1097-2765 ] ; 1997.

RBID : ISTEX:5A64CCDEB988BAAB8B68AA768CC76A177DA18F13

English descriptors

Teeft :
- Active site tyrosine, Adduct, Alkaline phosphatase, Biol, Ccctt, Chem, Cleavage, Cleavage product, Cleavage products, Cleavage reaction, Cleavage site, Covalent, Covalent adduct, Covalent adduct formation, Covalently, Cyclic, Cyclic phosphate, Denaturing polyacrylamide, Duplex, Enzyme, Ethanol, Ethanol precipitation, Eukaryotic, Eukaryotic type, Fmol, Free cleavage product, Human topoisomerase, Ligation, Molecular cell, Nucleic, Nucleic acid, Nucleic acids, Nucleotide, Petersen, Phosphatase, Phosphodiester, Pmol, Polyacrylamide, Precipitation, Reaction mixture, Reaction mixtures, Reaction products, Religation, Ribonuclease, Ribonuclease activity, Ribonucleoside, Ribonucleotides, Ribose sugar, Rnase, Rnase activity, Scissile, Scissile phosphate, Scissile phosphodiester, Scissile ribonucleotide, Scissile strand, Sekiguchi, Shuman, Single ribonucleoside, Single ribouridine substitution, Strand, Strand cleavage, Strand scission, Strand transfer reactions, Suicide substrate, Topoisomerase, Topoisomerase cleavage, Topoisomerase cleavage product, Topoisomerases, Transesterification, Transesterification reaction, Trna, Vaccinia, Vaccinia topoisomerase, Vaccinia virus, Westergaard.

Abstract

Abstract: Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein–DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3′-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2′ OH of the ribose sugar to yield a free 2′, 3′ cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.

Url:

https://api.istex.fr/ark:/67375/6H6-Z1MVRQ98-F/fulltext.pdf

DOI: 10.1016/S1097-2765(00)80010-6

Links to Exploration step

ISTEX:5A64CCDEB988BAAB8B68AA768CC76A177DA18F13

Le document en format XML

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<note type="content">Figure 1: DNApRNA Substrates Tandem DNApDNA and DNApRNA 36-mers were synthesized using vaccinia DNA ligase and then hybridized to a 36-mer DNA strand to form duplex molecules A–E. Ribonucleotides in italics. The pentapyrimidine recognition site for vaccinia topoisomerase is demarcated by the box. The site of covalent adduct formation is indicated by the arrow.</note>
<note type="content">Figure 2: Topoisomerase Is a Site-Specific Ribonuclease Cleavage reaction mixtures (20 μl) containing 50 mM Tris HCl (pH 8.0), 100 fmol of 32P-labeled substrates A–E as indicated, and 500 fmol of topoisomerase (plus) were incubated for 30 min at 37°C. Control mixtures lacked topoisomerase (minus). The reactions were quenched by adding SDS to 1%. The nucleic acid was ethanol-precipitated and the reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. An autoradiogram of the gel is shown.</note>
<note type="content">Figure 3: Topoisomerase Concentration Dependence of RNA Cleavage Reaction mixtures (20 μl) containing 50 mM Tris HCl (pH 8.0), NaCl as indicated, 100 fmol of 32P-labeled substrate D, and 0, 20, 50, 100, or 500 fmol of topoisomerase were incubated for 60 min at 37°C. The reactions were quenched by adding SDS to 1%. The nucleic acid was ethanol-precipitated and the reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. The extent of conversion of the labeled 36-mer strand to the free 20-mer cleavage product (fmol) was quantitated by scanning the gel with a Phosphorimager and is plotted as a function of input enzyme.</note>
<note type="content">Figure 4: Topoisomerase Cleavage of RNA Leaves a 2′, 3′ Cyclic Phosphate End The gel-purified 32P-labeled product of topoisomerase cleavage of substrate C (lane 1) was digested with alkaline phosphatase (lane 2), RNase A (lane 3), or RNase A followed by alkaline phosphatase (lane 4). The gel-purified 32P-labeled product of RNase A digestion of substrate C (lane 5) was digested with alkaline phosphatase (lane 6). The 32P-labeled 36-mer scissile strand of substrate C (lane 7) was digested with alkaline phosphatase (lane 8) to confirm that the phosphatase was not contaminated with ribonuclease.</note>
<note type="content">Figure 5: RNA Cleavage by Topoisomerase Requires the Active Site Tyrosine Reaction mixtures containing 100 fmol of substrate B or C and 500 fmol of wild-type topoisomerase or the Phe-274 active site mutant were incubated for 30 min at 37°C. The 32P-labeled reaction products were analyzed by denaturing polyacrylamide gel electrophoresis.</note>
<note type="content">Figure 6: Kinetic Analysis of Topoisomerase-Mediated RNA Cleavage Reaction mixtures containing (per 20 μl) 100 fmol of substrate D and 500 fmol of topoisomerase were incubated at 37°C. Aliquots (20 μl) were withdrawn at the times indicated and quenched immediately with SDS. The reaction products were recovered by ethanol precipitation and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. The gel was scanned with a phosphorimager. The extent of transfer of the 32P-labeled scissile strand to the topoisomerase to form the covalent adduct (which migrated just below the well) and the extent of formation of the free cleavage product (expressed as the percent of the total radioactivity in each species) are plotted as a function of time. The figure shows a line plot of the data.</note>
<note type="content">Figure 7: A Single Ribonucleoside at the Scissile Phosphate Is Sufficient for RNA Cleavage The structure of the ribouridine-substituted suicide substrate is shown. Reaction mixtures contained 0.5 pmol of suicide substrate (lane 1), 0.5 pmol of suicide substrate plus 2.5 pmol of topoisomerase (lane 2), 0.5 pmol of the CCCTU-containing 18-mer single strand (lane 3), 0.5 pmol of the CCCTU-containing 18-mer plus 2.5 pmol of topoisomerase (lane 4), or 0.5 pmol of the CCCTU-containing 18-mer plus 1 μg of RNase A (lane 5).</note>
<note type="content">Figure 8: Cleavage at a Single Ribonucleoside in Duplex DNA The structure of the ribouridine-substituted 30 bp equilibrium substrate is shown. A reaction mixture (100 μl) containing 50 mM Tris HCl (pH 8.0), 0.4 pmol of 30 bp substrate, and 5 pmol of topoisomerase was incubated at 37°C. Aliquots (10 μl) were withdrawn at the times indicated and quenched immediately in SDS. The samples were adjusted to 50% formamide and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. The gel was scanned with a phosphorimager. The extent of formation of the free cleavage product is plotted as a function of time.</note>
<note type="content">Figure 9: Reaction of Human Topoisomerase I with Deoxythymidine- and Ribouridine-Containing Scissile Strands The structure of the dimeric DNA cleavage substrate is shown with scissile phosphodiester Tp↓A indicated by arrows. The ribouridine-substituted substrate contains (rU)p↓A at the cleavage site. The reaction mixtures in lanes 1–3 contained 20 fmol of DNA substrate and 0 (lane 1), 50 (lane 2), or 100 ng (lane 3) of human topoisomerase I. The reaction products were digested with proteinase K, recovered by ethanol precipitation, and resolved by gel electrophoresis. Labeled products were visualized by autoradiography. The positions and sizes of oligonucleotide markers are indicated on the left. The reaction mixtures in lanes 4 and 5 contained 20 fmol of ribouridine-substituted substrate and 0 (lane 4) or 100 ng (lane 5) of human topoisomerase I. The reaction products were recovered by ethanol precipitation without proteinase K digestion. An aliquot of a ribonuclease A digest of the 32P-labeled ribo-substituted 30-mer scissile strand was electrophoresed in parallel (lane 6).</note>
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<abstract xml:lang="en"><p>Abstract: Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein–DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3′-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2′ OH of the ribose sugar to yield a free 2′, 3′ cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.</p>
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<ce:text>Corresponding author: Stewart Shuman, 212 639 7145 (phone), 212 717 3623 (fax)</ce:text>
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<ce:abstract-sec><ce:simple-para>Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein–DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3′-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2′ OH of the ribose sugar to yield a free 2′, 3′ cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.</ce:simple-para>
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<abstract lang="en">Abstract: Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein–DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3′-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2′ OH of the ribose sugar to yield a free 2′, 3′ cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.</abstract>
<note type="content">Section title: Article</note>
<note type="content">Figure 1: DNApRNA Substrates Tandem DNApDNA and DNApRNA 36-mers were synthesized using vaccinia DNA ligase and then hybridized to a 36-mer DNA strand to form duplex molecules A–E. Ribonucleotides in italics. The pentapyrimidine recognition site for vaccinia topoisomerase is demarcated by the box. The site of covalent adduct formation is indicated by the arrow.</note>
<note type="content">Figure 2: Topoisomerase Is a Site-Specific Ribonuclease Cleavage reaction mixtures (20 μl) containing 50 mM Tris HCl (pH 8.0), 100 fmol of 32P-labeled substrates A–E as indicated, and 500 fmol of topoisomerase (plus) were incubated for 30 min at 37°C. Control mixtures lacked topoisomerase (minus). The reactions were quenched by adding SDS to 1%. The nucleic acid was ethanol-precipitated and the reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. An autoradiogram of the gel is shown.</note>
<note type="content">Figure 3: Topoisomerase Concentration Dependence of RNA Cleavage Reaction mixtures (20 μl) containing 50 mM Tris HCl (pH 8.0), NaCl as indicated, 100 fmol of 32P-labeled substrate D, and 0, 20, 50, 100, or 500 fmol of topoisomerase were incubated for 60 min at 37°C. The reactions were quenched by adding SDS to 1%. The nucleic acid was ethanol-precipitated and the reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. The extent of conversion of the labeled 36-mer strand to the free 20-mer cleavage product (fmol) was quantitated by scanning the gel with a Phosphorimager and is plotted as a function of input enzyme.</note>
<note type="content">Figure 4: Topoisomerase Cleavage of RNA Leaves a 2′, 3′ Cyclic Phosphate End The gel-purified 32P-labeled product of topoisomerase cleavage of substrate C (lane 1) was digested with alkaline phosphatase (lane 2), RNase A (lane 3), or RNase A followed by alkaline phosphatase (lane 4). The gel-purified 32P-labeled product of RNase A digestion of substrate C (lane 5) was digested with alkaline phosphatase (lane 6). The 32P-labeled 36-mer scissile strand of substrate C (lane 7) was digested with alkaline phosphatase (lane 8) to confirm that the phosphatase was not contaminated with ribonuclease.</note>
<note type="content">Figure 5: RNA Cleavage by Topoisomerase Requires the Active Site Tyrosine Reaction mixtures containing 100 fmol of substrate B or C and 500 fmol of wild-type topoisomerase or the Phe-274 active site mutant were incubated for 30 min at 37°C. The 32P-labeled reaction products were analyzed by denaturing polyacrylamide gel electrophoresis.</note>
<note type="content">Figure 6: Kinetic Analysis of Topoisomerase-Mediated RNA Cleavage Reaction mixtures containing (per 20 μl) 100 fmol of substrate D and 500 fmol of topoisomerase were incubated at 37°C. Aliquots (20 μl) were withdrawn at the times indicated and quenched immediately with SDS. The reaction products were recovered by ethanol precipitation and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. The gel was scanned with a phosphorimager. The extent of transfer of the 32P-labeled scissile strand to the topoisomerase to form the covalent adduct (which migrated just below the well) and the extent of formation of the free cleavage product (expressed as the percent of the total radioactivity in each species) are plotted as a function of time. The figure shows a line plot of the data.</note>
<note type="content">Figure 7: A Single Ribonucleoside at the Scissile Phosphate Is Sufficient for RNA Cleavage The structure of the ribouridine-substituted suicide substrate is shown. Reaction mixtures contained 0.5 pmol of suicide substrate (lane 1), 0.5 pmol of suicide substrate plus 2.5 pmol of topoisomerase (lane 2), 0.5 pmol of the CCCTU-containing 18-mer single strand (lane 3), 0.5 pmol of the CCCTU-containing 18-mer plus 2.5 pmol of topoisomerase (lane 4), or 0.5 pmol of the CCCTU-containing 18-mer plus 1 μg of RNase A (lane 5).</note>
<note type="content">Figure 8: Cleavage at a Single Ribonucleoside in Duplex DNA The structure of the ribouridine-substituted 30 bp equilibrium substrate is shown. A reaction mixture (100 μl) containing 50 mM Tris HCl (pH 8.0), 0.4 pmol of 30 bp substrate, and 5 pmol of topoisomerase was incubated at 37°C. Aliquots (10 μl) were withdrawn at the times indicated and quenched immediately in SDS. The samples were adjusted to 50% formamide and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. The gel was scanned with a phosphorimager. The extent of formation of the free cleavage product is plotted as a function of time.</note>
<note type="content">Figure 9: Reaction of Human Topoisomerase I with Deoxythymidine- and Ribouridine-Containing Scissile Strands The structure of the dimeric DNA cleavage substrate is shown with scissile phosphodiester Tp↓A indicated by arrows. The ribouridine-substituted substrate contains (rU)p↓A at the cleavage site. The reaction mixtures in lanes 1–3 contained 20 fmol of DNA substrate and 0 (lane 1), 50 (lane 2), or 100 ng (lane 3) of human topoisomerase I. The reaction products were digested with proteinase K, recovered by ethanol precipitation, and resolved by gel electrophoresis. Labeled products were visualized by autoradiography. The positions and sizes of oligonucleotide markers are indicated on the left. The reaction mixtures in lanes 4 and 5 contained 20 fmol of ribouridine-substituted substrate and 0 (lane 4) or 100 ng (lane 5) of human topoisomerase I. The reaction products were recovered by ethanol precipitation without proteinase K digestion. An aliquot of a ribonuclease A digest of the 32P-labeled ribo-substituted 30-mer scissile strand was electrophoresed in parallel (lane 6).</note>
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Site-Specific Ribonuclease Activity of Eukaryotic DNA Topoisomerase I

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