"Since the two chains in our model are intertwined, it is essential for them to untwist if they are to separate.... Although it is difficult at the moment to see how these processes occur without everything getting tangled, we do not feel that this objection will be insuperable."
Watson and Crick, 1953
In the seminal paper quoted above, Drs. James D. Watson and Francis H.C. Crick correctly predicted that double-stranded DNA must be locally untwisted to enable gene transcription and chromosome replication. Scientists have known since the 1970s that this biological feat--akin to the familiar magic trick of unlinking two interlocked metal rings--is accomplished by a family of enzymes called topoisomerases.
Their critical role in cell division makes these enzymes prime targets for anticancer drugs. But until recently, scientists couldn't intentionally design such drugs because they knew so little about the enzymes. Now, the inner workings of one topoisomerase have been laid bare by a group of researchers led by NIGMS grantee Dr. James C. Wang, the Mallinckrodt Professor of Biochemistry and Molecular Biology at Harvard University.
In the January 18 issue of Nature, the scientists described the detailed, three-dimensional x-ray structure of yeast topoisomerase II, which is closely related to two forms of topoisomerase II in humans. In the April 30 issue of the Proceedings of the National Academy of Sciences (PNAS), the researchers explain how the yeast enzyme unlinks two interlocked rings of double-stranded DNA. In humans, topoisomerase II acts on long, tightly coiled linear DNA, separating newly replicated chromosomes prior to cell division.
"This work provides a molecular picture that basically proves the mechanism [by which the enzyme works]," said Dr. Wang. "This in turn has implications in the design of anticancer drugs."
The studies reveal that yeast topoisomerase II is heart-shaped, with a gaping hole in its center. Although it is unusual for a protein to have a hollow middle, even more surprising is the elaborate system of "hinges" and dramatic conformational changes the enzyme requires to perform its unlinking trick (see figure).
A model for the reaction catalyzed by topoisomerase II. Different domains of the enzyme are designated A', B', and C'. The two double-stranded DNA segments involved in the reaction (which can be part of much longer, linear DNA or interlocked, double-stranded rings) are represented by light and dark rods. In (1) topoisomerase II binds one segment of DNA, inducing a conformational change shown in (2). Upon binding ATP (represented by asterisks) and the other DNA segment (3), the enzyme undergoes a series of conformational changes: The A' parts of the enzyme separate, pulling apart the first DNA segment. At the same time, the second DNA segment is pushed through the severed first DNA segment into the central hole and the C' domains join together (4). The B' domain in front is transparent in (4) to permit visualization of the DNA behind it. The structure in brackets is a hypothetical intermediate. Many drugs that target topoisomerase II stabilize such an intermediate, preventing the enzyme from completing the reaction. In (5), the first DNA segment is resealed and the second DNA segment is released through the opening between the A' domains at the "bottom" of the enzyme. The A' domains then rejoin, ATP is converted to ADP and inorganic phosphate (Pi), and the enzyme returns to its form in (2). Although the enzyme would normally return to (1), the scientists designed their experiment so that the enzyme never released the first DNA segment.
Figure adapted from Nature, 18 January 1996, 379, p. 231 with permission.
Because drugs act on specific enzyme conformations, knowledge of these conformations has significant implications in drug design. "Topoisomerase is like several enzymes in one because its various conformations are so different," Dr. Wang said. "Each one is a potential drug target."
Many anticancer drugs, initially recognized for their ability to kill cells, have turned out to target topoisomerases, Dr. Wang said. Among these are doxorubicin (Adriamycin), etoposide, and mitoxantrone.
Because topoisomerase II is required during cell division, cancer cells, which divide wildly, are more sensitive to drugs that target topoisomerases than normal, more slowly dividing cells, Dr. Wang said. Part of the power of drugs that target topoisomerases is that they complement drugs that attack different cellular targets, and so can be used in aggressive combination chemotherapy.
"For unknown reasons, there are higher levels of topoisomerases in some cancer cells than in normal cells," Dr. Wang said. Many topoisomerase-targeting drugs "freeze" the enzyme while it is clutching two ends of a severed DNA strand before rejoining them (see figure). "Basically, what this type of drug does is to convert the normal enzyme into a DNA-damaging agent. Therefore, the more enzyme you have, the more potent the drug becomes." This is an unusual pharmaceutical mechanism, Dr. Wang said. "Usually, drugs inhibit a particular enzyme. These drugs convert the enzyme to a poison."
Such drugs have applications beyond chemotherapy. Antibiotics that target the bacterial form of topoisomerase II have been used since 1977. Now, by exploiting their developing knowledge of the differences between the human forms of topoisomerase and microbial forms, researchers are beginning to design antibiotic, antifungal, and antiparasitic agents that act specifically on the non-human forms of the enzymes.
Although he acknowledges that determining the structure of an enzyme is a far cry from marketing an acceptable human drug that efficiently targets that enzyme, Dr. Wang is hopeful that this relatively new approach to drug design will yield results.
"Structure-based design is still in its infancy, so there are people who are skeptical about it," Dr. Wang said. "My own bet is that this kind of work will get better and better, so sooner or later--hopefully sooner--we should see more drugs based on this approach."
Please acknowledge partial funding for this work from the National Institute of General Medical Sciences (NIGMS), a component of the National Institutes of Health that supports basic, non-disease-targeted research.
Berger JM, Gamblin SJ, Harrison SC, and Wang JC. Structure and Mechanism of DNA Topoisomerase II. Nature 1996;379:225-32.
Roca J, Berger JM, Harrison SC, and Wang JC. DNA Transport by a Type II Topoisomerase: Direct Evidence for a Two-Gate Mechanism. Proc. Natl. Acad. Sci. USA 1996;93:4057-62.
Dr. James C. Wang
To contact scientists available for comment, call the NIGMS Office of Communications and Public Liaison at (301) 496-7301.
Scientists already knew that topoisomerase II binds to double-stranded DNA, severs it, passes a second DNA double helix through the resulting gap, then reseals the original DNA. The question is, how? Two theories prevail. According to the "one-gate" theory, DNA enters and exits the enzyme's active site through the same "gate." According to the "two-gate" theory, DNA enters by one "gate" and exits, after the reaction, by a different "gate." The PNAS paper supports the "two-gate" theory.
The researchers used two approaches: to lock closed the putative "exit gate," using disulfide bonds, and to regulate opening of the putative "entry gate" with ATP or a non-hydrolyzable form of ATP called AMPPNP. Details available upon request from Alisa Zapp Machalek or Dr. James C. Wang.
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