High-resolution three-dimensional crystallography images of the binding
and cleavage core of type II topoisomerase (topo II) as it interacts with
DNA.
Topoisomerase has been called nature's magician because it can literally
pass one DNA segment through another. It is an enzyme that is divided
into two classes, type I and type II, depending on whether it cleaves one
or two strands of DNA during the catalytic cycle. Topo II cleaves a double-stranded
DNA, passes a second duplex through the break, and then immediately
repairs the broken strands. This enables topo II to control the topology
of DNA for chromosome segregation and disentanglement.
Using the exceptionally
bright and intense beams of x rays generated at ALS Beamline
8.3.1,
the researchers obtained high-resolution, three-dimensional crystallography
images of the DNA binding and cleavage core of a topo II enzyme
taken from yeast as it interacted with a segment of DNA. The images
revealed that topo II causes a sharp bend—150 degrees or more—in
the DNA segment at the point where it is cleaved. The near folding-in-half
of the DNA segment helps enable topo II to recognize where it should disentangle
DNA strands.
Large conformational changes in the topo II accompany
the DNA deformation, creating a bipartite catalytic site that positions
the DNA backbone near a reactive tyrosine and coordinated magnesium
ion. Remarkably, this configuration turns out to also closely resemble
the catalytic site of certain type I topoisomerases, which reinforces
the evolutionary link between what are otherwise structurally and
functionally distinct enzymes.
Based on the structural images, the
researchers believe that topo II employs a "two-gate" mechanism
to carry out its tasks. The upper domain of topo II opens to admit
a segment of DNA and transport it to the enzyme's core where the segment
is folded. A second DNA segment is then admitted and the upper domain gate
closes. This closing of the upper gate triggers the cleavage of
the bent DNA segment and the subsequent transport of the second
DNA segment through the break. When the gate in topo II's lower
domain swings open, the second DNA segment is released and the
cleaved DNA segment is reconnected. In many ways, the enzyme works
like a set of canal locks, opening and closing certain protein
interfaces, or gates, to control the passage of one DNA segment
through another without accidentally letting go of the DNA and
breaking the chromosome irreversibly.
In this two-gate model of topo II, the upper gate opens to admit a DNA
duplex, the G-segment, which is then transported to the enzyme's core where
it is bent. The admission of a second DNA duplex, the T-segment, causes
the G-segment to be cleaved. After the T-segment is transported through
the break, a lower gate opens for its release, causing the G-segment to
be reconnected. (Click on the image to see a Quick Time movie showing topo
II in action.)
To the credit of biochemists
and chemists, their discovery and refinement of anti-topo II drugs
have already made a remarkable therapeutic impact. Yet, all of the work
on these compounds has been done without a good picture of how type II topoisomerases
engage DNA. This new structural knowledge fills that hole, and
should be of significant help for guiding the development of future anti-topo
II drugs with improved efficacy. The researchers are now looking into producing
crystallographic images of topo II as it interacts with antibacterial
and anticancer drugs to determine the rules of engagement.
Research conducted by K.C. Dong and J.M. Berger (University of California,
Berkeley).
Research funding: National Cancer Institute and National Institutes of
Health. Operation of the ALS is supported by the U.S. Department of Energy,
Office of Basic Energy Sciences (BES).
Publication about this research: K.C. Dong and J.M. Berger, "Structural
basis for Gate-DNA recognition and bending by type IIA topoisomerases," Nature 450,
1201 (2007). |