New Mechanistic and Functional Insights into DNA Topoisomerases

Supplementary Data

• Download all Supplemental Material as a PDF. Includes Supplemental Figures 1-5 (also reproduced below), Supplemental Table 1, and references.

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  Supplemental Figure 1: TopIIA. Structure of type IIA topoisomerase. (a) A structural model of human Top2α (hTop2α). This hypothetical model was constructed based on the EM images of full-length type II enzymes, with the AMPPNP-bound ATPase and transducer domain (the N-gate, PDBid: 1ZXM,) on top and the DNA-bound cleavage core (PDBid: 4FM9) at bottom. No structural information is currently avaible for the C-terminal domain of eukaryotic Top2β and this region is omitted in this model. To highlight the dimeric assembly of the type IIA enzyme and the bipartite formation of the N-, DNA- and C-gate, one subunit is shown in gray and the other is colored by individual domains as indicated. (b) Orthogonal views of etoposide-stabilized cleavage complex of human Top2β (PDBid: 3QX3). The two etoposide molecules that insert into DNA cleavage sites (red arrowheads) are shown in yellow sticks. For both panels, DNA is shown in cartoon/surface presentation. The two active site tyrosine residues are shown with space-filling in red.

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  Supplemental Figure 2: Catalytic cycle of TopII. Outlines of the mechanisms of type II topoisomerase. (a) Schematic diagram for the proposed two-gate mechanism of type IIA enzyme. States 1 and 2 represent the quaternary conformations adopted by the apoenzyme, in which the N-gate is open and the cleavage core is in equilibrium between an open (State 1) and a closed form (State 2). Upon the binding of G-segment (State 3), the two ATPase domains move toward one another to half-close the N-gate, ready for the capture of T-segment. ATP-binding brings the N-gate to its full closure to entrap the T-segment and stimulates the unlocking of the DNA-gate (State 4), via the formation of double-strand break with 4-base pair overhangs (middle). The hydrolysis of one of the bound ATP conincides with passage of T-segment through the opened DNA-gate (State 5). Clousre of the DNA-gate allows the cleaved G-segment to be resealed and prompts the C-gate opening to facilitate the release of T-segment (State 6). Resetting of the enzyme is achieved by the second ATP hydrolysis. (b) Schematic diagram for the proposed two-gate mechanism of type IIB enzyme. The proposed catalytic cycles of the two classes of type II topoisomerases are highly similar. The G-segment enters the apoenzyme (State 1) through an opened N-gate (State 2). T-segment is captured upon the ATP-induced closure of the N-gate (State 3), and is transported through the cleaved G-segment upon the hydrolysis of one ATP (State 4). There are two major distinctions between type IIA and IIB topoisomerases: type IIB enzymes produce 2-base pair overhangs (middle); and the DNA- and C-gate are structurally merged in type IIB enzyme such that the release of T-segment conincides with its passage. Following G-segment religation, the second ATP hydrolysis takes place to reset the enzyme.

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  Supplemental Figure 3: TopIIB. Structure of type IIB topoisomerase. Ribbon representation of the structure of full-length Top6 from Methanosarcina mazei (PDBid: 2Q2E). One subunit is shown in gray and the other is colored by individual domains following the color code used in Supplementary Fig. 1.

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  Supplemental Figure 4: Top IB. Structure of type IB topoisomerase. Ribbon representation of the topotecan-stabilized cleavage complex of human Top1 (PDBid: 1K4T). The drug molecule that inserts into DNA cleavage site (red arrowhead) is shown in yellow sticks. The structure is colored by individual domains as indicated.

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  Supplemental Figure 5: Top IA. Structure of type IA topoisomerase. Ribbon representation of the cleavage complex of E. coli Top1A (PDBid: 3PX7). The structure is colored by individual domains following the color code used in Supplementary Fig. 1.