1. Topoisomerase Type II Structures
Comparison & Implications of the ’07 Berger & Dong Model

2. Topoisomerase: what is it?
Enzymes within cells that remove excessive supercoiling—and thereby torsional stress—while maintaining sufficient supercoiling to ensure a stable compact genome.
These enzymes are apparent in every cellular function involved with DNA—transcription, replication, recombination, and chromosome separation.
The enzymes complete their job in any of the above function by cleavage; that is, cutting.
Type I topoisomerase cut only 1 strand of DNA, Type II topoisomerase cut both (2) strands of DNA.

3. What’s Our Type? Type I Isomerase Type II Isomerase
lA: links to 5’-phosphate terminus, relaxes negatively supercoiled DNA, introduces transient breaks in ss region of DNA
IB: links 3’ terminus covalently to tyrosol group, relaxes both negatively and positively supercoiled DNA, segment can rotate with respect to protein to relieve torsional stress
Neither require ATP energy
Both cut only 1 strand of DNA
Type II Isomerase
IIA: found in eukaryotes, highly expressed in cancerous cells
IIB: structurally and biochemically distinct from Type IIA, found in archaea and some higher plants
Both require ATP energy to activate
Both perform a double-stranded cut of DNA

4. What’s Our Type? Type I Isomerase Type II Isomerase
lA: links to 5’-phosphate terminus, relaxes negatively supercoiled DNA, introduces transient breaks in ss region of DNA
IB: links 3’ terminus covalently to tyrosol group, relaxes both negatively and positively supercoiled DNA, segment can rotate with respect to protein to relieve torsional stress
Neither require ATP energy
Both cut only 1 strand of DNA
Type II Isomerase
IIA: found in eukaryotes, highly expressed in cancerous cells
IIB: structurally and biochemically distinct from Type IIA, found in archaea and some higher plants
Both require ATP energy to activate
Both perform a double-stranded cut of DNA

5. Topoisomerase Type IIA
Mechanism: A two-gate structure, Type II topoisomerases work by cleaving and opening one DNA duplex, passing a second duplex through the opening, and then resealing the break.

6. Topoisomerase Type IIA
Function: Type II topoisomerase performs a cycle of DNA cleavage, transport, and religation (coupled with ATP binding and hydrolysis) to preserve the topological invariant of the DNA
That is, it relaxes positively and negatively charged supercoiled DNA and performs cantenations/decantenations to prevent torsional stress and keep the cell’s compact genetic structure
This is an essential function for cell functionality and survival, and so all organisms have at least one type of Type II topoisomerase!

7. Berger & Dong 2007 Model Summary Conclusions Impact Limitations
Comparison to Previous Models
Berger et al Model 1996
Rybenkov et al Model 1997
Vologodskii et al Model 2001

8. Berger and Dong 2007 Model Summary
The molecular understanding of DNA binding to Topo II was examined
Crystallization of the Topo II-DNA complex was performed
Crystallography determines the arrangement of atoms in solids

9. Berger & Dong 2007 Model Summary Conclusions Impact Limitations
Comparison to Previous Models
Berger et al Model 1996
Rybenkov et al Model 1997
Vologodskii et al Model 2001

10. Berger and Dong 2007 Model Conclusions
Upon binding, Topo II distorts the DNA at ~150 degree global angle
The mechanism of this bend very similar to remodeling proteins
Along with the change in DNA’s conformation, Topo II undergoes a conformational change
This change forms a catalytic site for the ‘cutting’ reaction
Change in Top2’s conformation (shape)
Bending of DNA

11. Berger & Dong 2007 Model Summary Conclusions Impact Limitations
Comparison to Previous Models
Berger et al Model 1996
Rybenkov et al Model 1997
Vologodskii et al Model 2001

12. Berger and Dong 2007 Model
The researchers concluded that their findings:
Corroborated previous biochemical findings about Topo II
Established the structure as mechanism intermediate
Discovery of DNA cleavage controls
***Control of DNA topology
Is Topo II attracted to curvature OR does it cause the curvature? (Which came first, the chicken or the egg?)
Question is resolved….Topo II CAUSES curvature! (via bending)
This bending provides a means to decatenation and relaxation
Preferred binding highly curved DNA crossovers and loops in vivo probably arise from intrinsic deforms DNA

13. Berger & Dong 2007 Model Summary Conclusions Impact Limitations
Comparison to Previous Models
Berger et al Model 1996
Rybenkov et al Model 1997
Vologodskii et al Model 2001

14. Berger and Dong 2007 Model Limitations
Only a portion of Topo II was examined
Fragment used was ~720 aa long (residues )
Experiment performed in vitro
Model organism used was S. cerevisiae, or yeast (is structure/binding/bending conserved with other organisms?

15. Berger & Dong 2007 Model Summary Conclusions Impact Limitations
Comparison to Previous Models
Berger et al Model 1996
Rybenkov et al Model 1997
Vologodskii et al Model 2001

16. Berger Model 1996
1996 cartoon
One of the researchers of Dong/Berger model published a report on the Structure and Mechanism of Topo II 12 years ago
The Topo II examined was also S. cerevisiae of similar size to 2007 model
In 1996, researchers thought conformation changes occurred after passage of T-segment, not upon binding of G-segment
Bending of DNA upon binding with Topo II was not detected
2007 cartoon

17. Rybenkov Model 1997 Top2 bound to crossover
This study didn’t specifically look at the structure of Topo II, but it examined implications of its mechanism
Rybenkov et al concluded that Topo II doesn’t simply catalyze the passing of one DNA strand through another, changing its local conformation and topology, but it controls the global property of DNA as well
Suggested Topo II catalyzed the passage of one strand through another if resulting crossing angles were favorable
Suggested Topo II shortened DNA
Topo II binds three (3) DNA segments (crossover complex)
Top2 bound to crossover

18. Vologodskii Model 2001
Simulation of DNA
This study attempted to use its model to explain previous experimental data
Computer Analysis of enzyme performed
Discrete worm-like chain
Monte Carlo procedure simulated possible DNA conformations
Found that simplification of DNA topology by Topo II not possible without distortion of DNA
Suggested that Topo II create asymmetry in DNA upon binding
DNA distortion by Topo II

19. Applications of the Berger & Dong 2007 Model
Summary of Topoisomerase Type II pharmaceuticals
Existing limitations with current Topoisomerase Type II-focused medications
Increasing drug efficiency with Berger & Dong’s current Topoisomerase Type II model

20. Pharmaceuticals Summary
Mammalian type II topoisomerases are targets of anti-tumor drug classes
- aminoacridines, anthracenediones, ellipticines, and epipodophyllotoxins.
- many of them interact with DNA by an intercalative mode
Bacterial type II toposiomerases are targets of antibacterial gents
- quinolones and flouroquinolones.

21. DNA intercalators are used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells.
Ethidium intercalated between
two adenine-uracil base pairs.

22. How does drug work on DNA?
The drug ,localized precisely at the site of DNA cleavage, prevents topoisomerase from rejoining DNA breaks.
Cleavage complexes themselves are not sufficient to cause cell death.
Active cellular processes (transcription/replication) are necessary to transform cleavage complexes into cytotoxic lesions

23. Complex by protein-denaturant treatment-NaDodSO4/alkali
After binding to one segment of duplex DNA, a type II topoisomerase cleaves the duplex with a four-base stagger.
Model for the initial steps in the topoisomerase Il-catalyzed DNA strand-passing reaction. (A) Unbound topoisomerase. (B) Noncleavable complex. (C) Cleavable complex.

24. Current Limitations Multi-drug resistance
- Altered topoisomerase II complex activities
- results in decreased ability to form a stable ternary complex
- current measurements of topoisomerase II not reveal the relevant qualitative change

25. Current Limitations
Unrecognized cofactors may influence topoisomerase II activity and confound analyses.
Drug-topoisomerase II interactions and the precise defects in cells( altered activity) are not understood completely.

26. Pharmaceutical Impact
With the current Berger and Dong model for Topoisomerase Type II, we can:
Examine complex DNA and see if its geometry is changed by experimental drugs
Test inhibitors for Topoisomerase Type II to prevent DNA bending and kill the cell
Prevent rigidity in Topoisomerase Type II treatments due to built-up resistance

27. Further Research

28. Future Research Multicellular Confirmation Enzyme Shift
Is Berger and Dong’s model conserved when the experiment is repeated on multicellular organisms
Enzyme Shift
Following Berger and Dong’s model bending DNA, does the enzyme undergo another conformational change prior to cutting the DNA

29. Future Research Chemical basis of DNA cleavage
Benefits of unidirectional transport – not understood
Visualization of DNA cleavage

30. References - “Structure and mechanism of DNA topoisomerase II”
+ James Berger & Ken Dong; Nature, Vol. 379, Jan. 1996, pp. 225 – 232
- “Simplification of DNA Topology Below Equilibrium Values by Type II Topoisomerases”
+ Valentin Rybenkov et al; Science; Vol. 277; Aug. 1997; pp. 690 – 693
- “Mechanism of topology simplification by type II DNA topoisomers”
+ Alexander Vologodskii et al; PNAS; Vol. 98 No. 6; Mar. 2001; pp – 3049
- “An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Baccteriophage T4 Replication Fork in Vivo”
+ George Hong & Kenneth Kreuzer; Molecular and Cellular Biology; Vol 20 No. 2; Jan. 2000; pp
“Involvement of DNA topoisomerase II in the selective resistance of a mammalian cell mutant to DNA minor groove ligands: lingand-induced DNA—protein crosslinking and responses to topoisomerase poisons
+ Paul J. Smith et al; Carcinogenesis; Vol 11 No. 4; pp
“Multidrug Resistance Associated With Altered Topoisomerase II Activity—Topoisomerases II as Targets for Rational Drug Design”
+ Kenneth Cowan & Charles Marrow; National Cancer Institute; February 28, 1990.
“Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisomerase II on DNA by 4’-(9-acridinylamino)-methanesulfon-m-anisidide”
+ Eric M. Nelson et al; Proc. Natl. Acd. Sci. USA; Vol 81; Mar 1984; pp
“Design of DNA Intercalators To Overcome Topoisomerase II-Mediated Multidrug Resistance”
+ Bruce C. Baguley et al; Journal of the National Cancer Institute; Vol 82; Mar. 1990; pp