Polynucleotide Ligase Activity of Eukaryotic Topoisomerase I Stewart Shuman  Molecular Cell  Volume 1, Issue 5, Pages 741-748 (April 1998) DOI: 10.1016/S1097-2765(00)80073-8

Figure 1 Endoribonucleolytic Cleavage by Topoisomerase through a Covalent Intermediate See text for further details. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 2 Topoisomerase Cleavage at a Single Ribonucleoside in Duplex DNA The structure of the CCCTT(rU)-containing 30 bp substrate is shown. (A) Cleavage reaction mixtures (20 μl) containing 80 fmol of 32P-labeled substrate and 0, 1.6, 3.1, 6.2, 12.5, 25, 50, 100, or 250 fmol of topoisomerase were incubated for 60 min at 37°C. The reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. The extent of transfer of the 32P label from the input 30-mer scissile strand to the topoisomerase to form the covalent adduct, the extent of formation of the free 12-mer cleavage product, and the residual 30-mer scissile strand (expressed as the percent of the total radioactivity in each species) are plotted as a function of input enzyme. (B) Binding reaction mixtures (20 μl) containing 50 mM Tris–HCl (pH 8.0), 6% glycerol, 80 fmol of 32P-labeled 30-mer, and 0, 12.5, 25, 50, 100, 250, or 500 fmol of topoisomerase (lanes 1–7) were incubated for 5 min at 37°C. The samples were electrophoresed through a 6% nondenaturing polyacrylamide gel in 0.25 × TBE. An autoradiogram of the gel is shown. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 3 Rate of RNA Cleavage Reaction mixtures containing (per 20 μl) 80 fmol of 32P-labeled 30 bp substrate and either 1000 fmol (A) or 25 fmol (B) of topoisomerase were incubated at 37°C. Aliquots were withdrawn at the times indicated and quenched immediately with SDS. The reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. The extent of transfer of the 32P label from the input 30-mer scissile strand to the topoisomerase to form the covalent adduct, the extent of formation of the free 18-mer cleavage product, and the residual 30-mer scissile strand (expressed as the percent of the total radioactivity in each species) are plotted as a function of time. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 4 Cleavage of a CCCT(rU) Strand with a Short Leaving Group The structure of the CCCTT(rU)-containing 24-mer/30-mer substrate is shown. (A) Cleavage reaction mixtures (20 μl) containing 80 fmol of 32P-labeled substrate and 0, 6.2, 12.5, 25, 50, 100, 250, 500, or 1000 fmol of topoisomerase were incubated for 60 min at 37°C. The extent of transfer of the 32P label from the input 24-mer scissile strand to the topoisomerase to form the covalent adduct, the extent of formation of the free 18-mer cleavage product, and the residual 24-mer scissile strand (expressed as the percent of the total radioactivity in each species) are plotted as a function of input enzyme. (B and C) Reaction mixtures containing (per 20 μl) 80 fmol of 32P-labeled 24-mer/30-mer and either 1000 fmol (B) or 25 fmol (C) of topoisomerase were incubated at 37°C. Aliquots were withdrawn at the times indicated. The percents of the total radioactivity as 24-mer, 18-mer, and covalent adduct are plotted as a function of time. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 5 Polynucleotide Ligase Activity of Vaccinia Topoisomerase The structure of the singly nicked ligation substrate containing 2′,3′-cyclic phosphate and 5′-OH termini at the nick is shown in (A). The control substrate containing a 2′,3′-cyclic phosphate terminus, but no 5′-OH acceptor strand, is shown in (B). Ligation reaction mixtures (20 μl) containing 15 fmol of 32P-labeled substrate A or B, and 1 pmol of topoisomerase (+) were incubated for 35 min at 37°C. Control reactions lacked topoisomerase (−). The 32P-labeled reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. An autoradiogram of the gel is shown. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 6 Enzyme Dependence and Kinetic Analysis of Polynucleotide Ligation (A) Enzyme dependence. Ligase reaction mixtures (20 μl) containing 15 fmol of 32P-labeled nicked substrate and 0, 6.2, 12.5, 25, 50, 100, 250, 500, or 1000 fmol of topoisomerase were incubated for 30 min at 37°C. The 32P-labeled reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. The extent of strand joining (% of total label in the 36-mer product) is plotted as a function of input enzyme. (B) Kinetics. Reaction mixtures containing (per 20 μl) 15 fmol of 32P-labeled substrate and 1000 fmol of topoisomerase were incubated at 37°C. Aliquots were withdrawn at the times indicated. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 7 Ligase Activity of Topoisomerase Requires Catalytic Residues Arg-130 and Lys-167 Reaction mixtures (20 μl) containing 15 fmol of 32P-labeled nicked substrate and either wild-type (WT) topoisomerase (1.25, 2.5, or 5 pmol), K167A (0.62, 1.25, 2.5, or 5 pmol), or R130A (0.62, 1.25, 2.5, or 5 pmol) were incubated for 30 min at 37°C. A control reaction lacked topoisomerase (−). The 32P-labeled products were analyzed by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The upper panel shows the top of the gel with the covalent protein-DNA adduct (denoted by the asterisk) retained near the sample well. The lower panel shows the 32P-labeled 18-mer substrate and the 36-mer ligation product. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 8 Ligase Activity of Topoisomerase Does Not Require the Active Site Nucleophile Tyr-274 (A) Reaction mixtures (20 μl) containing 15 fmol of 32P-labeled nicked substrate and 0, 0.31, 0.62, 1.25, 2.5, 5, or 10 pmol of Y274F were incubated for 30 min at 37°C. The extent of strand joining (% of total label in the 36-mer product) is plotted as a function of input enzyme. (B) Reaction mixtures containing (per 20 μl) 15 fmol of 32P-labeled substrate and 5 pmol of Y274F were incubated at 37°C. Aliquots were withdrawn at the times indicated. Ligation is plotted as a function of incubation time. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)

Figure 9 Mechanism of Polynucleotide Ligation by Y274F See text for further details. Molecular Cell 1998 1, 741-748DOI: (10.1016/S1097-2765(00)80073-8)