Topoisomerase Type II
Structures
Comparison & Implications of the
’07 Berger & Dong Model
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.
What’s Our Type?
• Type I 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 doublestranded cut of DNA
What’s Our Type?
• Type I 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 doublestranded cut of DNA
Topoisomerase Type IIA
• Mechanism: A twogate structure, Type II
topoisomerases work
by cleaving and
opening one DNA
duplex, passing a
second duplex
through the opening,
and then resealing
the break.
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!
Berger & Dong 2007 Model
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Summary
Conclusions
Impact
Limitations
Comparison to Previous Models
– Berger et al Model 1996
– Rybenkov et al Model 1997
– Vologodskii et al Model 2001
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
Berger & Dong 2007 Model
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Summary
Conclusions
Impact
Limitations
Comparison to Previous Models
– Berger et al Model 1996
– Rybenkov et al Model 1997
– Vologodskii et al Model 2001
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
Bending of DNA
Change in
Top2’s
conformation
(shape)
Berger & Dong 2007 Model
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•
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Summary
Conclusions
Impact
Limitations
Comparison to Previous Models
– Berger et al Model 1996
– Rybenkov et al Model 1997
– Vologodskii et al Model 2001
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 sites @ highly curved DNA crossovers and
loops in vivo probably arise from intrinsic deforms DNA
Berger & Dong 2007 Model
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•
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Summary
Conclusions
Impact
Limitations
Comparison to Previous Models
– Berger et al Model 1996
– Rybenkov et al Model 1997
– Vologodskii et al Model 2001
Berger and Dong 2007 Model
Limitations
• Only a portion of Topo II was examined
– Fragment used was ~720 aa long (residues 4081177)
• Experiment performed in vitro
• Model organism used was S. cerevisiae, or
yeast (is structure/binding/bending conserved
with other organisms?
Berger & Dong 2007 Model
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•
•
•
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Summary
Conclusions
Impact
Limitations
Comparison to Previous Models
– Berger et al Model 1996
– Rybenkov et al Model 1997
– Vologodskii et al Model 2001
Berger Model 1996
• 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 Tsegment, not upon binding of
G-segment
• Bending of DNA upon binding
with Topo II was not detected
1996 cartoon
2007 cartoon
Rybenkov Model 1997
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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
Vologodskii Model 2001
• 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
Simulation of DNA
DNA distortion by Topo II
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
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.
• DNA intercalators are
used in
chemotherapeutic
treatment to inhibit
DNA replication in
rapidly growing
cancer cells.
Ethidium intercalated between
two adenine-uracil base pairs.
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
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.
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
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.
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
Further Research
Future Research
• Multicellular Confirmation
– 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
Future Research
• Chemical basis of
DNA cleavage
• Benefits of
unidirectional
transport – not
understood
• Visualization of DNA
cleavage
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. 3045 – 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.594-603
- “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.659-665
- “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.1361-1365
- “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.398-402