
Topoisomerase II Structure Suggests Novel DNA Cleavage Mechanism Print

Type II topoisomerases are molecular machines that regulate DNA supercoiling and separate interlocked chromosomes. These enzymes are also exploited clinically as targets of antibiotics and anticancer therapeutics. Researchers at ALS Beamline 8.3.1 imaged type II topoisomerase’s ordinarily short-lived state in which it is linked to a DNA’s nucleic acid segment through its active site tyrosine, cleaving the DNA. Details of this molecular model provide evidence for the chemical mechanism by which type II topoisomerases (topo IIs) and a related topo family (topo IA) accomplish DNA cleavage. The structure also reveals how the enzyme avoids dissociating when DNA is cleaved, preventing the aberrant formation of mutagenic genomic lesions.

A Cellular Achilles Heel

Because DNA is a long, doubly-wound polymer, processes such as chromosome compaction and replication naturally lead to DNA overwinding, underwinding, and tangling. Topoisomerases are essential to managing these processes; however, the DNA cleavage chemistry used to resolve these altered DNA structures can also operate as a weak link that can be exploited to kill cells—in a good way.

A variety of small molecules known as “poisons” have been found to interfere with topoisomerase-mediated resealing of DNA following cleavage, leading to the formation of DNA nicks and double-stranded DNA breaks that promote cell death. Several poisons have been successfully exploited in the clinic as antibiotics such as ciprofloxacin (against bacterial topoisomerases) and as anticancer agents (e.g., etoposide and doxorubicin).

Unfortunately, many of the drugs that exploit topoisomerases have deleterious side effects or are losing potency due to the evolutionary emergence of drug-resistant enzymes. Understanding in atomic detail how existing drugs associate with their topoisomerase targets will spur the development of next-generation therapeutics that circumvent such shortcomings. The research done here provides a view of the DNA cleavage state of a type II topoisomerase similar to that found in humans, providing a key step toward imaging the enzyme bound both to DNA and anticancer compounds.

Left: Crystallographic model of yeast topoisomerase II (cylinders) bound and covalently linked to DNA (blue). Right: Electron density showing the covalent link between the catalytic tyrosine and DNA.

Type II topoisomerases disentangle DNA by creating a double-stranded break in one DNA segment, passing a second DNA segment through the break. Although structural and biochemical efforts have illuminated key aspects of topo II’s mechanism, an understanding of the chemistry and control of DNA cleavage has long remained elusive.

Proposed topoisomerase cleavage mechanism. The general base (B:) and acid (HA) are unknown, but may be metal-associated waters. Metal A and R781 stabilize the transition state, while metal B and H736 help anchor the -1/-2 phosphate.

To approach this problem, researchers used a chemically modified suicide-DNA substrate to ensnare the normally transient DNA-cleaved state of topo II. Collection of diffraction data from crystals of this topoisomerase•DNA complex at ALS Beamline 8.3.1 resulted in a high-resolution (2.5 Å), three-dimensional image of the assembly’s structure. Clear electron density gave evidence for a covalent link between DNA and the catalytic tyrosine of the topo II responsible for cleaving DNA, as well as for two metal ions associated with the active site.

Metal ions are used widely to aid enzyme catalysis by lowering the energy of the transition state. Interestingly, the organization of metal ions in the covalent topoisomerase•DNA complex differs from that of other well-studied systems: only one ion is positioned to assist with transition state chemistry, while the other plays a structural role in anchoring DNA. Thus, the structure reveals that type II topoisomerases employ a novel variation of canonical two-metal mechanisms. Comparison of the active site with other enzymes further shows that this approach is used by type IA topoisomerases as well, establishing an evolutionary link between the two enzyme families.

Left: Superposition of noncovalent (gray) and cleavage (green) complexes between topo II and DNA reveals how C-gate opening and closure is linked to active-site status. The connection from the active-site tyrosines to the “legs” is colored blue/magenta. Upper right: Upward movement of the active-site tyrosine upon becoming attached to the DNA. Lower right: Concomitant inward movement of the legs through a conserved salt-bridge network.

All topoisomerases transiently break the phosphodiester backbone of DNA during formation of the covalent phosphotyrosine intermediate. This reaction is required for topoisomerases to disentangle chromosomes and manage genome topology, but can lead to potentially cytotoxic or mutagenic DNA strand breaks if mismanaged.

The newly-imaged structure of the topoisomerase•DNA complex unexpectedly shows how this DNA breakage is coordinated with the association and dissociation of key protein interfaces that regulate the passage of DNA strands through said breaks and prevent accidental chromosome fragmentation.

A major difference between a previous, noncovalent DNA-topo II structure and this present model is that a key dimer interface of the enzyme switches from a dissociated (“open”) to an associated (“closed”) state. Comparing the structures reveals the molecular basis for this transition: formation of a covalent attachment with DNA pulls the catalytic tyrosine toward the duplex from its position in the uncleaved state by ~6 Å. This movement tugs on a linker element—like the drawstring on a latch—that engages a set of a-helical “legs” to close the dimer interface. Remarkably, this transition allows the status of the active site to be communicated over 50 Å away to a gating element required for DNA passage.

Such tight control over DNA cleavage and intersubunit associations helps explain how the fidelity of DNA breakage and rejoining by type II topoisomerases is maximized to protect the genome from the accidental formation of toxic DNA lesions during supercoiling management and chromosome disentanglement.



Research conducted by B. Schmidt and J. Berger (University of California, Berkeley); A. Burgin (Emerald BioStructures); and J. Deweese and N. Osheroff (Vanderbilt University School of Medicine).


Publication about this research: B. Schmidt, A. Burgin, J. Deweese, N. Osheroff, and J. Berger, “A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases," Nature 465, 641 (2010).

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