S. cerevisiae Topoisomerase II-DNA-AMPPNP Complex

Jonathan Pang '17 and Kenyatta Viel '17

Contents:

I. Introduction

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Saccharomyces cerevisiae topoisomerase II (topo II), a cellular enzyme, removes and adds DNA tangles, knots, and supercoils. The removal of a DNA tangle involves a DNA gate segment and a transient segment6. Topo II hydrolyzes ATP in order to perform a transesterification reaction between two tyrosine residues in the active site and a pair of phosphodiester bonds in DNA.


Cleavage of DNA by tyrosines 782 and 784.


The cleavage of the phosphodiester bonds creates an opening in the gate DNA segment, and allows the transient DNA segment to pass through. After the two segments of double-stranded DNA have been separated, another transesterification reaction occurs, where topo II re-ligates the DNA, and the covalent bonds between topo II and the DNA are broken2. On the left, topo II is complexed with DNA and the nonhydrolyzable ATP analog, AMPPNP. Two key structural features of topo II include the ATPase domain in the N gate, where ATP hydrolysis occurs, and the DNA gate, which contains the DNA cleavage site2.

II. General Structure


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Topo II is a homodimer. Each monomer of topo II is made up of an ATPase domain, a DNA binding/cleavage core (subfragments A' and B'), and a C terminal tail region2. The ATPase domain can be further divided into the GHKL and Transducer subdomains1. Subfragment A' is comprised of two domains. The first is the CAP-like domain (residues 682-820), which contains tyrosine 782 and tyrosine 784, the residues that covalently bond to the 5' end of the cleaved DNA. The second domain (residues 874-972) is made up of two beta sheets and two alpha helices. Subfragment B' is made up of two alpha/beta domains2. The C terminal tail is nonconserved across species, and its purpose is not fully understood. Removal of the 260 residues that make up the C terminus does not affect the function of topo II4.

III. ATP Binding


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In our model, AMPPNP, a nonhydrolyzable ATP analog, binds the ATPase domain. This is necessary to observe conformational changes to the structure of topo II when ATP is bound1. "Structure of AMPPNP" The presence of ATP causes a conformational cascade where the two ATPase domains dimerize. The ATP binding site is formed by the GHKL and Transducer subdomains. In the GHKL subdomain, asparagine 70 and lysine 147 form hydrogen bonds with the alpha phosphate group of ATP, and serine 127 and asparagine 129 form hydrogen bonds with the beta phosphate group of ATP. The glycine 365 and lysine 367 residues of the Transducer subdomain form hydrogen bonds with the gamma phosphate group of ATP. Two water molecules and two magnesium mediate the ATP hydrolysis reaction. The carbonyl oxygen of asparagine 70 participates in an octahedral coordination of the magnesium ion with the oxygens of the alpha and beta phosphates3


IV. DNA Binding


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Once ATP binds the ATPase domain, the A' subfragments separate to form a semicircular groove that narrows down to the the active site containing tyrosine 782 and tyrosine 784. The positive electrostatic potential of topo II attracts the negatively charged sugar-phosphate backbone of DNA to this groove. When ATP binds, the CAP-like domain of subfragment A' changes conformation to make contact with the DNA4. More specifically, the residues lysine 700, tyrosine 734, serine 740, alanine 780 of the helix-turn-helix motif in the CAP-like domain contact the 3' end of the duplex DNA. Tryptophan 908 also interacts with DNA, and isoleucine 833 intercalates between base pairs in DNA. These contacts stabilize the DNA and allow it to interact with active site tyrosines 782 and 7842.

Lysines 334-338 make significant electrostatic contacts with DNA. This is important because these lysine residues are part of the ATPase domain, so these contacts represent a direct connection between the ATPase domain and DNA. However, the specific bonds formed between the lysines in the ATPase domain and DNA are not yet known. Interactions between the ATPase domain and DNA are a topic of interest for future research1.

V. DNA Cleavage


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After the DNA has binded to the A' subfragment, the active-site tyrosines participate in a transesterification reaction to cleave DNA and again to re-ligate it. The oxygen on tyrosines 782 acts as a nucleophile, and attacks the phosphate in the DNA backbone5. A two-metal mechanism stabilizes and cleaves the DNA. The identity of the divalent metal ions involved is not yet known. However, it is known that in this arrangement, the metal ions form electrostatic interactions with the oxygen in the phosphate backbone of DNA. One metal ion forms electrostatic interactions with the oxygens of aspartic acid 526 and aspartic acid 528, and the phosphate adjacent to the phosphate that is cleaved. The other metal ion forms interactions with the oxygens on aspartic acid 526, glutamic acid 449, and the oxygens on the phosphate that is attacked by the active-site tyrosine. Histidine 736 and arginine 781 form hydrogen bonds directly to the phosphate groups in order to stabilize the DNA. In the transesterification reaction that ultimately breaks the DNA backbone, a base activates the oxygen of the active-site tyrosine so that it can act as a nucleophile. The active-site tyrosine then attacks the phosphate. One of the phosphate oxygens acts as a leaving group, and is stabilized by an acid. The acids and bases involved have not yet been determined, but they are likely water molecules6. DNA cleavage cannot proceed until ATP hydrolysis has occurred. Since our model was crystallized using a nonhydrolyzable ATP analog, The DNA shown at the left has not yet been cleaved.

VI. Implications


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Entanglements in DNA occur during replication, repair, and recombination. These chromosomal entanglements can lead to cell death if chromosomes are not able to separate during cell division2. For this reason, research concerning topoisomerase II has serious implications for cancer treatment. For example, the anticancer agent ICRF-187 binds to topo II and causes a conformational change that renders the enzyme inactive. Specifically, ICRF-187 targets topo II that has already formed bonds with ATP and DNA. It binds to the 14 residue drug-binding pocket, and bridges the two ATPase domains together3. The stabilization of the ATPase domains essentially converts topo II into a clamp on the DNA that prevents it from being disentangled or released.

VII. References

1. Schmidt B.H., Osheroff N., and Berger J.M. 2012. Structure of a topoisomerase II/ DNA/nucleotide complex reveals a new control mechanism for ATPase activity. Nature Structural & Molecular Biology, 19(11): 1147 - 1154.

2. Berger J.M., Gamblin S.J., Harrison S.C., and Wang J.C. 1996. Structure and mechanism of DNA topoisomerase II. Nature, 379: 225 - 232.

3. Classen S., Olland S., and Berger J.M. 2003. Structure of the topoisomerase II ATPase region and its mechanism of inhibition by the chemotherapeutic agent ICRF-187.Proc. Natl. Acad. Sci. U.S.A., 100(19): 10629 - 10634.

4. Berger J.M. and Wang J.C. 1996. Recent developments in DNA topoisomerase II structure and mechanism. Curr. Opin. Struct. Biol. 6(1): 84 - 90.

5.Deweese J.E., and Osheroff N. 2009. The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing.Nucl. Acids Res. 37(3):738-748.

6.Schmidt B.H., Burgin A. B., Deweese J.E., Osheroff N., Berger J.M. 2010. A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases.Nature, 465(7298): 641 - 644.

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