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Topo II: An Enzyme Target for Antibacterial and Cancer Drugs Print

The veil has finally been lifted on an enzyme that is critical to the process of DNA transcription and replication and is a prime target of antibacterial and anticancer drugs. Researchers at Berkeley Lab and the University of California, Berkeley, have produced the first three-dimensional structural images of a DNA-bound type II topoisomerase (topo II) that is responsible for untangling coiled strands of the chromosome during cell division. Preventing topo II from disentangling a cell's DNA is fatal to the cell, which is why drugs that target topo II serve as agents against bacterial infections and some forms of cancer. This first ever structural image of topo II should help in the development of future antibacterial and anticancer drugs that are even more effective and carry fewer potential side effects.

Detangling DNA

If the DNA in a single set of human chromosomes is stretched out and joined together, it measures about two meters in length. To be packed within the tiny confines of a cell's nucleus, all of this double-stranded DNA must be tightly bundled by a process known as supercoiling. During cell division, these coils of DNA give rise to knots and jumbles that must be unlinked. Failure to properly do so can give rise to chromosome breaks, which in turn can lead to genome instabilities and cell death. The topo II enzyme performs this unraveling for DNA in the cell. Antibacterial and anticancer drugs that target topo IIs and other topoisomerases, such as the quinolone family of antibiotics (of which the commonly-used ciprofloxacin is a member), work by preventing the enzymes from completing their tasks. Because the targeting of these drugs has not been optimal, there have sometimes been side effects that pose their own health risks. The structural studies conducted by Dong and Berger should serve as a useful platform for future efforts to understand the chemical basis of DNA cleavage and for efforts to understand and improve anti-topoisomerase therapeutics.

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. (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).