REPORTS
References and Notes
1. M. L. Yarbrough et al., Science 323, 269 (2009);
10.1126/science.1166382.
2. C. A. Worby et al., Mol. Cell 34, 93 (2009).
3. M. P. Müller et al., Science 329, 946 (2010);
10.1126/science.1192276.
4. A. W. Ensminger, R. R. Isberg, Curr. Opin. Microbiol. 12,
67 (2009).
5. T. Murata et al., Nat. Cell Biol. 8, 971 (2006).
6. M. P. Machner, R. R. Isberg, Dev. Cell 11, 47 (2006).
7. M. P. Machner, R. R. Isberg, Science 318, 974 (2007);
10.1126/science.1149121.
8. A. Ingmundson, A. Delprato, D. G. Lambright, C. R. Roy,
Nature 450, 365 (2007).
9. I. Derré, R. R. Isberg, Infect. Immun. 72, 3048 (2004).
10. J. C. Kagan, M. P. Stein, M. Pypaert, C. R. Roy, J. Exp.
Med. 199, 1201 (2004).
11. C. R. Roy, S. Mukherjee, Sci. Signal. 2, pe14 (2009).
12. M. Mantel, H. Holzer, Proc. Natl. Acad. Sci. U.S.A. 65,
660 (1970).
13. V. Spirin, L. A. Mirny, Proc. Natl. Acad. Sci. U.S.A. 100,
12123 (2003).
14. Z. Q. Luo, R. R. Isberg, Proc. Natl. Acad. Sci. U.S.A. 101,
841 (2004).
15. W. B. Anderson, E. R. Stadtman, Biochem. Biophys. Res.
Commun. 41, 704 (1970).
Cilia-Like Beating of Active
Microtubule Bundles
Timothy Sanchez,1 David Welch,2 Daniela Nicastro,3 Zvonimir Dogic1*
The mechanism that drives the regular beating of individual cilia and flagella, as well as dense
ciliary fields, remains unclear. We describe a minimal model system, composed of microtubules and
molecular motors, which self-assemble into active bundles exhibiting beating patterns reminiscent
of those found in eukaryotic cilia and flagella. These observations suggest that hundreds of
molecular motors, acting within an elastic microtubule bundle, spontaneously synchronize their
activity to generate large-scale oscillations. Furthermore, we also demonstrate that densely packed,
actively bending bundles spontaneously synchronize their beating patterns to produce collective
behavior similar to metachronal waves observed in ciliary fields. The simple in vitro system
described here could provide insights into beating of isolated eukaryotic cilia and flagella, as
well as their synchronization in dense ciliary fields.
ew behavior frequently emerges as complex structures are assembled from simpler
components. In biology, an example of
such hierarchical organization is the axoneme—
the core structure of eukaryotic cilia and flagella
(1). Important building blocks of axonemes are
dynein motors, which convert energy from adenosine triphosphate (ATP) hydrolysis into linear
movement along a microtubule (MT) track (2).
In each axoneme, dyneins are assembled onto
a scaffold of nine cylindrically arranged doublet
MTs. Dyneins are bound to one doublet MT
through an ATP-insensitive cargo-binding site
while moving in an ATP-dependent manner
along the neighboring MT. This causes sliding
of neighboring doublet MTs against each other,
which is thought to be subsequently converted
N
1
Department of Physics, Brandeis University, 415 South Street,
Waltham, MA 02454, USA. 2Graduate Program in Biophysics
and Structural Biology, Brandeis University, 415 South Street,
Waltham, MA 02454, USA. 3Department of Biology, Brandeis
University, 415 South Street, Waltham, MA 02454, USA.
*To whom correspondence should be addressed. E-mail:
zdogic@brandeis.edu
456
into bending by elastic connectors that restrict
intermicrotubule sliding (3, 4). The activity of
thousands of dyneins within each axoneme is
regulated to form oscillatory beating patterns, an
emergent behavior that is qualitatively different
from the linearly moving isolated dynein motors.
However, the exact mechanism that causes the
formation of self-sustained beating patterns of
isolated axonemes remains unclear (5). At the
next level of hierarchy, axonemes are the essential building blocks of dense ciliary fields, where
thousands of individual cilia beat in a tightly coordinated fashion. In these arrays, the neighboring cilia are not in identical states of their beat
cycle but are offset by a well-defined phase shift,
generating regions of higher density that travel
as metachronal waves and are capable of either
propelling entire cells or moving fluid across the
surface of various tissues. Metachronal waves are
critical for a variety of processes, including the
motility of ciliated protists, mucus clearance by
airway cilia, and the determination of the left/right
symmetry during embryonic development in humans (6, 7). Defective cilia are associated with
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16. H. Plutner et al., J. Cell Biol. 115, 31 (1991).
17. R. A. Bacon, A. Salminen, H. Ruohola, P. Novick,
S. Ferro-Novick, J. Cell Biol. 109, 1015 (1989).
18. E. J. Tisdale, J. R. Bourne, R. Khosravi-Far, C. J. Der,
W. E. Balch, J. Cell Biol. 119, 749 (1992).
19. I. Derré, R. R. Isberg, Infect. Immun. 73, 4370
(2005).
20. B. Byrne, M. S. Swanson, Infect. Immun. 66, 3029
(1998).
Acknowledgments: We thank G. Storz, S. Gottesman,
J. Bonifacino, J. Neunuebel, and M. Barzik for critical
reading and insightful discussion and R. Isberg and
V. Losick for kindly providing reagents. This work was
supported by the Intramural Research Program of the NIH.
Supporting Online Material
www.sciencemag.org/cgi/content/full/science.1207193/DC1
Materials and Methods
Figs. S1 to S17
Tables S1 and S2
References (21–27)
17 January 2011; accepted 7 June 2011
Published online 16 June 2011;
10.1126/science.1207193
severe human diseases (6). Previous studies of
axoneme beating have primarily used a top-down
approach by deconstructing a functional organelle
in order to identify the functional components required for beating (8–11). Here, using a bottom-up
approach, we identify a few essential components
required for an assembly of active MT bundles, a
simple biological oscillator whose beating patterns
are reminiscent of isolated cilia. This minimal in
vitro system is similar to previously proposed
theoretical models of cilia and flagella (12, 13).
Furthermore, we also demonstrate that dense fields
of interacting active MT bundles exhibit synchronous beating behavior similar to metachronal
waves observed in ciliary fields (14, 15).
The assembly of oscillatory active bundles
requires three main components (Fig. 1A). The
first two, biotin-labeled kinesin motors bound
into clusters through multimeric streptavidin and
taxol-stabilized MTs, spontaneously organize
into asterlike structures, reminiscent of mitotic
spindles (16, 17). The kinesin clusters simultaneously bind and walk along two neighboring MTs, resulting in their relative displacement
(“inter-MT sliding”). Addition of a third component, nonadsorbing polymer polyethylene glycol (PEG), alters the behavior of the system and
leads to the assembly of oscillating bundles. PEG
induces attractive interactions between MTs
through the depletion mechanism (fig. S1), leading to their bundling (18). Having MTs side by
side greatly increases the probability of kinesin
clusters simultaneously binding and walking
along neighboring MTs. The relative motion of
MTs in these bundles depends on their polarity;
whereas motor clusters generate displacement
between MTs of opposite polarity, no displacement is induced between MTs with the same
polarity (19).
To assemble self-oscillating MT bundles, we
flowed the MT/kinesin/PEG mixture into a flow
chamber coated with a polymer brush to avoid
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effector protein SidM, which mediates release of
Rab1 from its chaperone GDP dissociation inhibitor (RabGDI) (7, 8), Rab1 activation through
GDP/GTP exchange, and AMPylation. AMPylated
GTP-Rab1 is locked in the active conformation,
which prevents its premature inactivation by host
cell GAPs such as TBC1D20. AMPylated Rab1
could promote docking and fusion of secretory
vesicles with LCVs until L. pneumophila translocates the de-AMPylase SidD into host cells.
SidD catalyzes removal of AMP from GTP-Rab1,
thereby enabling Rab1 inactivation by the GAP
LepB and subsequent membrane extraction of
GDP-Rab1 by RabGDI.
The identification of SidD as the Rab1 deAMPylase not only provides the missing link in a
complex chain of Rab1 modulation events during
L. pneumophila infection, but also presents an
intriguing example for how prokaryotic and eukaryotic cells could regulate protein function
through transient AMPylation.
REPORTS
edges of the flow chamber or partially trapped
under air bubbles, and thus separated from the
bulk of the contracting structures. Once separated,
these bundles exhibited uniform large-scale beat-
Fig. 1. A minimal system of MTs, molecular motors, and depleting polymer assembles into actively
beating MT bundles. (A) Schematic illustration of all components required for assembly of active bundles.
(B) A sequence of images illustrating the beating pattern of an active bundle over one beat cycle. Scale
bar, 30 mm. (C) The conformation of the bending MT bundle indicates a fairly symmetric beating pattern
that is reminiscent of those found in cilia and flagella. (D) Sequence of images showing one beating cycle
of a flagellum from Chlamydomonas reinhardtii. Scale bar, 5 mm.
Fig. 2. A short MT bound to a longer filament by
the depletion force exhibits thermally driven sliding
motion. (A) A time-lapse observation of two sliding
MTs over a period of 15 min. Scale bar, 3 mm. At
this depletant concentration, unbinding events are
very rare. (B) A trajectory of a shorter MT diffusing while bound to the longer MT. The position is measured
relative to the end point of a longer MT. (C) MSD of the relative diffusion between the two MTs. The
subdiffusive behavior indicates viscoelastic coupling between the two filaments. The MSD data are
averaged for six filament pairs in which shorter filaments were between 3 and 4 mm long. In each
case, the dynamics were observed for anywhere between 10 and 30 min.
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VOL 333
ing patterns (Fig. 1, B and C, fig. S2, and movies
S1 to S3). To ensure that background MTs did not
interfere with the beating patterns, in some experiments we displaced the unbound MT/kinesin
mixture with a buffer containing only ATP, PEG,
and kinesin clusters (but no MTs). In these samples, the attached bundles continued to beat for
more than 1 hour. The primary factor that determines the beating frequency is the length of the
bundle, which can range from less than 10 mm to
more than 100 mm, with longer bundles beating
with longer periods (fig. S3). We hypothesize that
active bundles contain MTs with mixed polarity
and that the oscillatory beating is driven by the
activity of kinesin clusters that induce interfilament sliding. At the same time, the osmotic pressure due to PEG keeps the bundle together and,
in combination with the attachment at the base,
transforms the sliding motion into local bending, producing large-scale beating patterns.
How does the minimal system of active bundles compare with biological axonemes? Although
both have a scaffold composed of MTs, cilia and
flagella are highly ordered structures that contain more than 650 different proteins (8). In contrast, active bundles are disorganized structures
of varying size that only contain MTs and kinesin
clusters. Active MT bundles exhibit oscillations
only when attached to a fixed boundary, similarly
to axonemes that are attached to the cell cytoskeleton through the basal body (21). Additionally, both active bundles and axonemes contain
molecular motors that drive interfilament sliding.
However, in axonemes, dynein motors are permanently attached to one doublet MT (in an
ATP-insensitive manner), while walking on a
neighboring doublet in an ATP-dependent manner (4). In contrast, the in vitro MT bundles are
driven by kinesin clusters, which considerably
differ from axonemal dyneins in size and direction, as well as other aspects (22), including that
kinesin clusters continuously associate and dissociate from active bundles because they lack
permanent attachment sites to MTs.
Another requirement for the generation of
active oscillations in both systems is the presence
of condensing agents that arrange MTs into bundles and also might restrict MT sliding. In axonemes, specialized structures, such as the nexin
link, cross-link the nine MT doublets (11). It has
been suggested that the nexin binds, dissociates,
and rebinds neighboring MT doublets, thus allowing them to slide past each other over a significant distance while maintaining a well-defined
separation (23). In depletion-driven bundling, it
is not obvious if individual MTs slide freely past
each other or remain fixed in a more static structure due to large interfilament friction. The latter
was found to be the case for depletion-driven
bundling of actin filaments (24, 25). Using a recently developed method, we investigated the
dynamics of MT bundles confined to quasi–twodimensional (2D) chambers and found that MTs
slide freely past each other, indicating a relatively
low interfilament friction (Fig. 2, A and B, and
22 JULY 2011
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motility due to surface-bound motors (20). In the
bulk of the sample, the active MT network contracted into asterlike structures (16, 17); however,
a number of bundles remained attached to the
457
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458
(movie S10). The qualitative behavior of this selfassembled active system at an air-water interface
is similar to that of biological ciliary fields (com-
pare Fig. 3, C and E). In native systems, however,
metachronal waves travel typically over large regions with the same handedness (Fig. 3E).
Fig. 3. Propagation of metachronal waves
A
in a densely packed array of active MT bundles. (A) Interface of an air bubble covered
with a dense monolayer of beating bundles
(see also movie S7). The bundles are trapped
at their bases between the glass and an
air bubble. Regions of the edge that are
covered with a sufficiently high density
B
D
of active MT bundles exhibit methachronal
traveling waves. The length of bundles is
~10 mm. Scale bar, 40 mm. (B) Time sequence (25-s time lapse) of one section
from (A) shows local propagation of metachronal waves. Scale bar, 20 mm. (C) Image
of a ciliary field on the cell surface of the
ciliate Opalina ranarum, which generates
traveling metachronal waves. Scale bar,
20 mm. (D) A kymograph of the fluorescence
E
intensity represents the local MT density
of the bundle field, with higher densities
displayed in red and lower densities in
blue. Easily distinguishable metachronal
waves travel in either a clockwise or counterclockwise direction at different regions of
interface. Low surface coverage at the top
of the interface results in asynchronous beating of active bundles. Dashed red line in
C
(A) indicates the position at which the kymograph was taken. (E) A kymograph of the
ciliate field shown in (C) is presented for
comparison. Dashed red line in (C) indicates
the position at which the kymograph was taken. [With permission from IWF gGMBH, images in (C) and (E)
are extracted from (33).]
Fig. 4. Quantitative analysis of the spatial and temporal properties of a travelling metachronal wave. (A)
A 3D plot of the correlation function of the density, r, is plotted as a function of spatial separation (D x)
and temporal time lag (Dt). (B) The spatial correlation function at zero time lag (Dt = 0) corresponds to the
red slice of the 3D surface plot in (A). The functional form indicates a periodic structure with a wavelength
of 54 mm. (C) The temporal autocorrelation plot corresponds to the blue slice of the surface plot in (A) at
zero spatial separation (D x = 0). The periodicity of this correlation highlights the time propagation of the
density wave, as each point becomes anticorrelated with itself when 1/2 of a wavelength has passed and
correlated once again when a full wavelength has passed.
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movie S4) (24). We measured the mean square
displacement (MSD) between two MTs as a function of time (Fig. 2C). Purely viscous coupling
would result in an MSD curve that increases
linearly over time with a slope of 1 on a log-log
plot (red curve in Fig. 2C). However, the measured MSD curves exhibited subdiffusive behavior, indicating that the coupling between MTs
has weak elastic as well as a viscous component
(black curve in Fig. 2C). In active bundles, an additional source of interfilament friction might
arise from the motor proteins themselves (26).
The relative importance of viscous and elastic
interactions in MT bundles for the formation of
beating patterns will have to be examined in
more detail in future studies. Interestingly, the
above-described four requirements for beating
of active bundles, i.e., (i) microtubule tracks, (ii)
motor complexes interacting with at least two
microtubules, (iii) boundary attachment, and (iv)
linkages permitting interfilament sliding, are almost identical to the main ingredients of previously proposed theoretical models of cilia and
flagella (12, 13).
A major difference between active bundles
and biological cilia and flagella is the significantly
slower beating frequency observed in the in vitro
system. In light of the marked differences in
length, width, motors, and MT organization, this
difference is not too surprising. A likely factor
contributing to the slower beating is the arrangement of the motors on the MTs. In cilia and
flagella, the motors are densely packed along
the doublets [20 motors per 100 nm (11)], and
they are fixed to one MT while walking on the
neighboring one, whereas in the in vitro system,
motors continuously walk, associate, and dissociate from both MTs.
In addition to exploring the behavior of isolated active MT bundles, our model system also
assembles into dense arrays, enabling us to study
the emergent behavior that arises from interactions between actively beating bundles (movies
S5 to S8). At sufficiently high density, active
bundles spontaneously synchronize to generate
metachronal waves similar to those observed in
native ciliary fields. Although the base of each
active MT bundle remains stationary, successive
phase shifts in their beat cycles generate regions
of higher MT bundle density that spontaneously
propagate along the interface at a well-defined
velocity (Fig. 3 and movie S7). Along an airwater interface, several regions of synchronous
beating could be distinguished, in which metachronal waves traveled in either a clockwise or
counterclockwise direction as seen in a kymograph in Fig. 3D. In a few cases, collective
metachronal waves were capable of transporting passive material (movie S9), similar to the
transport of food particles by ciliated protists or
mucus by ciliated epithelia. Although all active bundles were lodged onto the coverslip at
the edge of an air-water interface, bundles were
observed in focus at z positions away from the
chamber surface, indicating 3D beating patterns
REPORTS
References and Notes
1. D. Bray, Cell Movements: From Molecular to Motility
(Garland Publishing, New York, 2000).
2. S. L. Reck-Peterson et al., Cell 126, 335 (2006).
3. P. Satir, J. Cell Biol. 39, 77 (1968).
4. K. E. Summers, I. R. Gibbons, Proc. Natl. Acad. Sci. U.S.A.
68, 3092 (1971).
5. C. J. Brokaw, Cell Motil. Cytoskeleton 66, 425 (2009).
6. B. A. Afzelius, J. Pathol. 204, 470 (2004).
7. Y. Okada, S. Takeda, Y. Tanaka, J. C. I. Belmonte,
N. Hirokawa, Cell 121, 633 (2005).
8. G. J. Pazour, N. Agrin, J. Leszyk, G. B. Witman, J. Cell Biol.
170, 103 (2005).
9. V. Stolc, M. P. Samanta, W. Tongprasit, W. F. Marshall,
Proc. Natl. Acad. Sci. U.S.A. 102, 3703 (2005).
10. E. E. Dymek, E. F. Smith, J. Cell Biol. 179, 515 (2007).
11. T. Heuser, M. Raytchev, J. Krell, M. E. Porter, D. Nicastro,
J. Cell Biol. 187, 921 (2009).
12. F. Jülicher, J. Prost, Phys. Rev. Lett. 78, 4510 (1997).
13. S. Camalet, F. Jülicher, J. Prost, Phys. Rev. Lett. 82, 1590
(1999).
14. B. Guirao, J. F. Joanny, Biophys. J. 92, 1900 (2007).
15. S. Gueron, K. Levit-Gurevich, Proc. Natl. Acad. Sci. U.S.A.
96, 12240 (1999).
16. F. J. Nédélec, T. Surrey, A. C. Maggs, S. Leibler, Nature
389, 305 (1997).
17. T. Surrey, F. Nedelec, S. Leibler, E. Karsenti, Science 292,
1167 (2001).
18. D. J. Needleman et al., Phys. Rev. Lett. 93, 198104
(2004).
19. C. Hentrich, T. Surrey, J. Cell Biol. 189, 465 (2010).
20. Materials and methods are available as supporting
material on Science Online.
21. B. H. Gibbons, I. R. Gibbons, J. Cell Biol. 54, 75 (1972).
22. R. D. Vale, Cell 112, 467 (2003).
23. I. Minoura, T. Yagi, R. Kamiya, Cell Struct. Funct. 24, 27
(1999).
24. A. W. C. Lau, A. Prasad, Z. Dogic, EPL 87, 48006
(2009).
25. M. M. Claessens, M. Bathe, E. Frey, A. R. Bausch,
Nat. Mater. 5, 748 (2006).
Structural Basis of Type II
Topoisomerase Inhibition by the
Anticancer Drug Etoposide
Chyuan-Chuan Wu,1 Tsai-Kun Li,2,3 Lynn Farh,4 Li-Ying Lin,1,5 Te-Sheng Lin,1,5 Yu-Jen Yu,1
Tien-Jui Yen,1 Chia-Wang Chiang,1,5 Nei-Li Chan1,5*
Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently
cleaving both strands of a DNA duplex to form a cleavage complex through which another
DNA segment can be transported. Several widely prescribed anticancer drugs increase the
population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA
breakage and death of cancer cells. We present the crystal structure of a large fragment of
human TOP2b complexed to DNA and to the anticancer drug etoposide to reveal structural
details of drug-induced stabilization of a cleavage complex. The interplay between the protein,
the DNA, and the drug explains the structure-activity relations of etoposide derivatives and
the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions
provides information applicable for developing an isoform-specific TOP2-targeting strategy.
ype II topoisomerases (TOP2s) alter DNA
topology and play roles in replication, transcription, recombination, and chromosome
condensation and segregation (1, 2). These twofold symmetric enzymes transiently cleave a pair
of opposing phosphodiester bonds four base
pairs apart, which generates a TOP2-DNA cleavage
complex. Passage of a second DNA segment through
this enzyme-bridged “DNA gate” and its resealing complete the topological change of the DNA
T
(3, 4). TOP2’s DNA cleavage activity is a doubleedged sword; failure to reseal the enzyme-mediated
DNA break can lead to cell death (5). Several potent anticancer drugs and antibiotics exploit this
harmful aspect of TOP2 and promote the formation of cytotoxic DNA lesions by increasing
the steady-state level of cleavage complexes (6–9).
However, despite the extensive use of TOP2targeting drugs in anticancer chemotherapy, the
lack of three-dimensional structures of any drug-
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VOL 333
26. V. Bormuth, V. Varga, J. Howard, E. Schäffer, Science
325, 870 (2009).
27. P. Y. Plaçais, M. Balland, T. Guérin, J. F. Joanny,
P. Martin, Phys. Rev. Lett. 103, 158102 (2009).
28. T. Guérin, J. Prost, P. Martin, J. F. Joanny, Curr. Opin. Cell
Biol. 22, 14 (2010).
29. F. Gittes, E. Meyhöfer, S. Baek, J. Howard, Biophys. J. 70,
418 (1996).
30. C. P. Brangwynne, G. H. Koenderink, F. C. Mackintosh,
D. A. Weitz, Phys. Rev. Lett. 100, 118104 (2008).
31. R. Dreyfus et al., Nature 437, 862 (2005).
32. M. Vilfan et al., Proc. Natl. Acad. Sci. U.S.A. 107, 1844
(2010).
33. E. Horstmann, IWF movie “C792/1959”: Flimmerepithel
(Institut fuer den Wissenschaftlichen Film, Goettingen
1964).
Acknowledgments: We acknowledge useful discussions with
S. Fraden and M. Hagan. We are grateful to J. Chung
and J. Gelles for their help in purification of microtubules
and kinesin and also acknowledge D. Chen and E. Peck
for images of beating Chlamydomonas reinhardtii.
We are thankful to the IWF for their permission to use
images of Opalina ranarum (Fig. 3C). This work was
supported by funding from the W. M. Keck Foundation,
National Science Foundation (DMR-MRSEC-0820492),
and National Institutes of Health (5K25GM85613 to
Z.D. and 5R01GM083122 to D.N.).
Downloaded from www.sciencemag.org on July 25, 2011
The metachronal waves of the artificial system were quantitatively analyzed in Fig. 4. The
3D spatial-temporal correlation function shown in
Fig. 4A indicates that the wavelength of the metachronal waves was 54 mm, the temporal frequency
is 4.9 × 10−3 Hz, and the velocity is 0.27 mm/sec.
By comparison, the ciliary field of the Opalina
ranarum produces metachronal waves with a spatial wavelength of 17 mm, a temporal frequency
of 3 Hz, and a velocity of 51 mm/sec. It has been
proposed that the highly coordinated behavior of
ciliary fields is not controlled by chemical signaling pathways but merely arises from hydrodynamic
coupling between neighboring cilia (14, 15). Our
observation of metachronal waves in a simplified
in vitro system supports this hypothesis.
In conclusion, we have demonstrated that a
simple nonequilibrium system, composed of MTs
and molecular motors, spontaneously synchronizes
on multiple levels of hierarchy, with qualitatively
new functions emerging at each hierarchical level.
This system should provide insight into the mechanisms that drive the oscillations of biological
nanomachines (27–30), while simultaneously
enabling assembly of active materials capable of
fluid transport and swimming motion (31, 32).
Supporting Online Material
www.sciencemag.org/cgi/content/full/333/6041/456/DC1
Materials and Methods
Figs. S1 to S3
References
Movies S1 to S10
8 February 2011; accepted 7 June 2011
10.1126/science.1203963
stabilized cleavage complexes has left the structural bases of drug actions and resistance largely
unresolved and has hampered the development
of isoform-specific TOP2-targeting agents (10).
To these ends, we determined the high-resolution
crystal structure of the DNA-binding and cleavage core of the human TOP2 b isoform (residues
445 to 1201; hereinafter abbreviated hTOP2bcore)
in complex with DNA and the anticancer drug
etoposide.
A ternary cleavage complex was prepared by
mixing purified hTOP2bcore with a 20–base pair
DNA duplex and etoposide (Fig. 1, A and B).
The DNA substrate contained a preferred 5′C↓NNNNG-3′ cleavage site (the arrow) in the
middle, flanked by nucleotides matching a deduced semiconsensus sequence (table S1). The
crystal structure of the etoposide-stabilized cleavage complex was determined at 2.16 Å resolution
(table S2) (11). The asymmetric unit comprised
one DNA duplex enclosed symmetrically by the
1
Institute of Biochemistry and Molecular Biology, College of
Medicine, National Taiwan University, Taipei City 100, Taiwan.
2
Department and Graduate Institute of Microbiology, College
of Medicine, National Taiwan University, Taipei City 100, Taiwan.
3
Center for Biotechnology, National Taiwan University, Taipei
City 106, Taiwan. 4Department of Chemical Biology, National
Pingtung University of Education, Pingtung, Pingtung County
900, Taiwan. 5Institute of Biochemistry, College of Life Sciences,
National Chung Hsing University, Taichung 402, Taiwan.
*To whom correspondence should be addressed. E-mail:
nlchan@ntu.edu.tw
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