1. The Mechanism of Type IA Topoisomerase-Mediated DNA Topological Transformations 
Zhiyu Li, Alfonso Mondragón, Russell J DiGate 
Molecular Cell 
Volume 7, Issue 2, Pages (February 2001)
DOI: /S (01)

2. Figure 1
Schematic Diagram of the Proposed Mechanism of Relaxation and Catenation/Decatenation Catalyzed by Type IA DNA Topoisomerases
Type IA DNA topoisomerases catalyze several different topological rearrangements of DNA. The reactions can be divided into (A) intramolecular and (B) intermolecular reactions.
(A) DNA topoisomerase-catalyzed relaxation of supercoiled DNA substrates has been proposed to occur by the enzyme (gray toroid with a yellow single-stranded binding region) binding to a small, denatured region within the supercoiled substrate (red/blue molecule) (a), followed by nicking of the scissile strand (b). The enzyme catalyzes a strand passage event (c), followed by resealing of the nicked strand (d). The enzyme releases the DNA (e) or begins another catalytic cycle. The reactions always proceed toward relaxation of the DNA.
(B) DNA topoisomerase-catalyzed catenation or decatenation of DNA proceeds via similar intermediates as described above (a–e), except that the scissile (shown in green) and passed strands (shown in red/blue) are present in two independent molecules. The passed strand can be either single or double stranded. In all cases of type IA-mediated catalysis, the scissile strand must be single stranded in the region of the active site (which only encompasses 5–10 bases), but may be double stranded at all other points. The reaction pathway is reversible, and can result in the catenation or decatenation of the two DNA molecules (shown by double arrows)
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Design of a Type IA DNA Topoisomerase with an Interdomain Disulfide Bond to Restrict Conformational Freedom
(A) Schematic diagram of E. coli DNA topoisomerase III based on the crystallographic structure (Mondragón and DiGate, 1999). Residues I299 and A494 of E. coli DNA topoisomerase III (indicated in green) were changed to cysteines by in vitro mutagenesis. Since the backbone carbon of I299 and A494 are located at the proper distance (7–8 Å) from one another and the side chain carbons face toward each other, this pair of cysteines can form a disulfide bond under oxidizing conditions. The four domains of the enzyme are labeled. The active site region (red) and the single-stranded DNA binding groove (yellow) are also indicated.
(B) Experimental protocol for trapping DNA/protein intermediates. A type IA DNA topoisomerase, capable of forming a disulfide bond linking domains III and IV (cysteine residues are indicated as green dots), is incubated with double-stranded DNA followed by addition of oxidized glutathione (GSSG). The formation of a disulfide bond (linked green dots) traps the DNA within the cavity. Reduction of the disulfide bond with dithiothreitol (DTT) returns conformational freedom to the molecule and allows the DNA to escape
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Supercoiled DNA Relaxation Activity of the Disulfide-Containing Topo III Is Stimulated by DTT and Inhibited by Oxidized Glutathione
(A) Topo III and Topo III-DS are active in the presence of a reducing agent. Relaxation assays were performed in the presence of increasing amounts of dithiothreitol. Lanes 1–4 contained 0.5 pmol of Topo III, lanes 5–8 contained 2.3 pmol of Topo III-DS, and lane 9 contained no protein. Lanes 1–4 and 5–8 contained increasing concentrations of DTT (0, 0.08, 0.4, and 2 mM). The reaction products were electrophoresed through a 1% agarose gel, and the DNA was visualized by staining with ethidium bromide. O.C.: open circular relaxed DNA; S.C.: supercoiled circular DNA.
(B) Topo III is active but Topo III-DS is inactive in the presence of an oxidizing agent. Relaxation assays, containing 0.4 mM DTT, were performed in the presence of increasing amounts of oxidized glutathione. Lanes 1–4 contained 0.5 pmol of Topo III, lanes 5–8 contained 2.3 pmol of Topo III-DS, and lane 9 contained no protein. Lanes 1–4 and 5–8 contained increasing concentrations of oxidized glutathione (0, 0.08, 0.4, and 2 mM). The reaction products were electrophoresed through a 1% agarose gel, and the DNA was visualized by staining with ethidium bromide. O.C.: open, circular relaxed DNA; S.C.: supercoiled circular DNA
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Filter Binding Assay of Topo III and the Disulfide-Containing Topo III with Different DNA Substrates
Topo III-DS(Y328F) and Topo III(Y328F) were tested for the ability to trap circular and linear double-stranded DNA and circular single-stranded DNA. DNA binding assays were performed as described (see Experimental Procedures). Identical reactions were filtered through a nitrocellulose membrane in the absence (red bars) or presence (green bars) of DTT. Only the enzyme capable of forming a disulfide bond trapped DNA, indicating that DNA could be retained inside the central hole of the enzyme. Topo III-DS(Y328F) trapped 112 fmol of supercoiled circular DNA, 51 fmol of relaxed circular DNA, and 27 fmol of single-stranded circular DNA on the filter
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Topo III Possesses at Least Two Independent DNA Binding Domains
To test whether Topo III could simultaneously bind DNA in the central hole and in the single-stranded DNA binding region, DNA was first trapped in the protein, followed by addition of a radiolabeled single-stranded oligonucleotide. To create the complex, DTT was added first to allow entrance to the protein, followed by glutathione addition to trap the DNA. Lane 2 clearly shows that when circular DNA is trapped inside the enzyme, it is still capable of forming a complex with single-stranded DNA. Binding assays contained 6 pmol of Topo III(Y328F) (lane 1), 6 pmol of Topo III-DS(Y328F) (lanes 2–5), or no protein (lane 6). DTT (for a final concentration of 1.25 mM), oxidized glutathione (for a final concentration of 10 mM), and supercoiled plasmid DNA (250 μg) were added to each reaction as indicated and described (see Experimental Procedures). The products were separated on a 1% TBE agarose gel containing 1 mM MgCl2 and visualized by autoradiography. The location of the DNA topoisomerases was accomplished by staining with ethidium bromide. The positions of open, circular relaxed DNA (O.C.), supercoiled DNA (S.C.), and linear (Lin.) are indicated. Topo III-single-stranded DNA complexes are not visible due to their fast mobility
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 Topo III Cannot Trap Linear Double-Stranded DNA Substrates
Closed circular DNA trapped inside Topo III-DS escapes when the DNA is linearized, suggesting that the protein can slide off the DNA due to weak protein–DNA interaction. Topo III-DS(Y328F) was incubated with circular, double-stranded DNA followed by addition of oxidized glutathione to trap the DNA. The reaction was split in two and either buffer (−) or HindIII restriction endonuclease (+) was added to the reactions. Both reactions were incubated at 37°C, the products were separated as described in Figure 5, and visualized by autoradiography. The location of the topoisomerases was accomplished by staining with ethidium bromide. S.C.: supercoiled circular DNA, Lin.: linear double-stranded DNA
Molecular Cell 2001 7, DOI: ( /S (01) )