Site-Specific Ribonuclease Activity of Eukaryotic DNA Topoisomerase I JoAnn Sekiguchi, Stewart Shuman  Molecular Cell  Volume 1, Issue 1, Pages 89-97 (December 1997) DOI: 10.1016/S1097-2765(00)80010-6

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. Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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. Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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. Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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. Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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. Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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. Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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). Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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. Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

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). Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)

Figure 10 Mechanism of RNA Cleavage by Type IB Topoisomerase Molecular Cell 1997 1, 89-97DOI: (10.1016/S1097-2765(00)80010-6)