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. 2001 Mar 13;98(6):3045-9.
doi: 10.1073/pnas.061029098.

Mechanism of topology simplification by type II DNA topoisomerases

A V Vologodskii  1 , W Zhang, V V Rybenkov, A A Podtelezhnikov, D Subramanian, J D Griffith, N R Cozzarelli
Affiliations

Mechanism of topology simplification by type II DNA topoisomerases

A V Vologodskii et al. Proc Natl Acad Sci U S A. .

Abstract

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

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Figures

Figure 1
Figure 1
Test of topology change by strand passage. For each case where a hairpin G segment (red) was juxtaposed with a potential T segment, the topology of the new conformation resulting from strand passage was calculated. To perform the test, we replaced the actual G segment by a bypass (pink) of the T segment. (A) For a hairpin G segment, the bypass was obtained by a 180° rotation of the hairpin. (B) For a straight G segment, the bypass was a four-segment loop directed toward the potential T segment.
Figure 2
Figure 2
Model of type II topoisomerase action. The enzyme (green) bends a G segment of DNA (red) into a hairpin. The entrance gate for the T segment of DNA (yellow) is inside the hairpin. Thus, the T segment can pass through the G segment only from inside to outside the hairpin.
Figure 3
Figure 3
Typical simulated conformation of a knotted DNA with a hairpin G segment (red). Another segment of the 7-kb model chain is inside the hairpin in this conformation, which was selected from the set generated by a Metropolis Monte Carlo procedure.
Figure 4
Figure 4
The effect of topoisomerase IV binding on the efficiency of DNA cyclization. (A) Gel electrophoretic separation of ligation products obtained in the absence and presence of topoisomerase IV for a 190-bp DNA. The results after the indicated times of ligation are shown. The bands are circular monomers (CM) and dimers (CD), and linear monomers (M), dimers (LD), and trimers (LT). (B) j Factors for eight DNA fragments in the absence (●) and presence (○) of topoisomerase IV. Error bars (standard deviation) smaller than the symbols are not shown. Solid lines represent a theoretical fit based on a Monte Carlo simulation using a DNA helical repeat of 10.55 bp, a persistence length of 40 nm, and a DNA torsional rigidity of 2 × 10−19 erg⋅cm (1 erg = 0.1 mJ). Enzyme binding was modeled by wrapping the model chain around a cylinder 14 nm in diameter with an angle α = 130° (see Inset for the angle definition).
Figure 5
Figure 5
Visualization of type II topoisomerase molecules bound to relaxed and (−) supercoiled DNA. pBR322 DNA was incubated with yeast topoisomerase II (A, B, D) or E. coli topoisomerase IV (C, E), as described in the text. The DNA was relaxed in A and B and supercoiled in C–E. The complex in C is an enlargement of a molecule in E. After incubation, the samples were prepared for EM by fixation and rotary shadowcasting with tungsten. Images are shown in reverse contrast. [Bar = 0.33 kb of DNA (A–C) and 1.0 kb of DNA (D, E).]
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