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Interdomain Communication in DNA Topoisomerase II

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Interdomain Communication in DNA Topoisomerase II THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 33, pp. 23395–23404, August 18, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Interdomain Communication in DNA Topoisomerase II DNA BINDING AND ENZYME ACTIVATION*□S Received for publication, May 1, 2006, and in revised form, June 16, 2006 Published, JBC Papers in Press, June 16, 2006, DOI 10.1074/jbc.M604119200 Felix Mueller-Planitz and Daniel Herschlag1 From the Department of Biochemistry, School of Medicine, Stanford University, Stanford, California 94305-5307 Topoisomerase II catalyzes the ATP-dependent transport of a clamps the ATPase domains around it (step 2). A transient dou- DNA segment (T-DNA) through a transient double strand break ble strand break is created in the G-DNA by two active site in another DNA segment (G-DNA). A fundamental mechanistic tyrosines that covalently attach to a 5Ј-phosphoryl group on question is how the individual steps in this process are coordi- each DNA strand. This break in the G-segment widens and acts nated. We probed communication between the DNA binding as a gate through which the T-segment is transported (step 3), a sites and the individual enzymatic activities, ATP hydrolysis, step that is coupled to hydrolysis of ATP (4, 5). The cleaved and DNA cleavage. We employed short DNA duplexes to con- G-DNA is religated, and the T-segment is released from the trol occupancy at the two binding sites of wild-type enzyme and enzyme (step 4). Downloaded from a variant with a G-DNA site mutation. The DNA concentration The catalytic functions of molecular machines, such as topo- dependence of ATP hydrolysis and a fluorescence anisotropy isomerase II, require the coordination of conformational assay provided thermodynamic information about DNA bind- changes. Such coordination can only be achieved if the regions ing. The results suggest that G-DNA binds with higher affinity of the enzyme that carry out the individual reaction steps can than T-DNA. Enzyme with only G-DNA bound is competent to energetically communicate with one another. How these indi- http://www.jbc.org/ cleave DNA, indicating that T-DNA is dispensable for DNA vidual steps modulate one another and how they are integrated cleavage. The ATPase activity of enzyme bound solely to into the overall reaction mechanism is therefore central to G-DNA is partially stimulated. Full stimulation requires bind- understanding molecular machines. ing of T-DNA. Both DNA binding sites therefore signal to the Several aspects of the intramolecular communication in ATPase domains. The results support and extend current mech- topoisomerase II have been established. For example, upon at Stanford University on February 12, 2016 anistic models for topoisomerase II-catalyzed DNA transport binding of ATP, the two ATPase domains of the enzyme self- and provide a framework for future mechanistic dissection. associate (6–8) by exchanging an N-terminal “strap” motif that wraps around the ATP binding pocket of the neighboring sub- unit (9), demonstrating physical communication between the DNA topoisomerase II is a molecular machine that ATPase domains. DNA cleavage is stimulated in the presence resolves topological problems generated during DNA repli- of ATP, indicating that the ATPase and the DNA cleavage cation, transcription, recombination, chromosome segrega- domains allosterically interact (4, 10, 11). However, the physical tion, and chromosome condensation (1). Topoisomerase II basis for the signaling between these domains is not known. has attracted attention not only for its importance during Although many aspects of the catalytic mechanism and cellular processes. It serves as a model system to mechanis- interdomain communication of topoisomerase II have been tically dissect allosteric, intramolecular communication of a established, fundamental questions about the intramolecular complex molecular machine. signaling mediating the coordinated DNA transport of topo- Biochemical and structural studies have suggested a isomerase II remain unresolved. It has been suggested, for mechanism for the overall catalytic mechanism of topo- example, that the enzyme must interact with the T-DNA before isomerase II (Fig. 1) (2, 3). According to this model, the DNA bound at the G-DNA site can be cleaved (19). However, enzyme binds to two different DNA segments. One segment, topoisomerase II with its ATPase gate “locked” was able to termed the gate or G-DNA, binds to the cleavage/religation cleave linear DNA (6). This result is consistent with a require- domain (Fig. 1, step 1). The other DNA segment, termed the ment of T-DNA binding for DNA cleavage only if the DNA can transported or T-DNA, binds and is trapped inside the enzyme thread into the enzyme to bind at both the T- and the G-DNA when ATP binding induces a conformational change that binding site (26). Similar uncertainty exists regarding the influence of the DNA molecules on the other enzymatic activity, ATP hydroly- * This work was supported by Boehringer Ingelheim Fonds (to F. M.-P.) and by National Institutes of Health Grant GM64798 (to D. H.). The costs of publi- sis. It is well established that the presence of DNA is sensed by cation of this article were defrayed in part by the payment of page charges. the ATPase domains, since the ATPase activity increases in This article must therefore be hereby marked “advertisement” in accord- response to DNA binding (see, for example, Refs. 4, 12, and 13), ance with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains but it has remained controversial whether binding the G-DNA supplemental Schemes 1–4, Figs. S1–S7, and Table S1. at the DNA cleavage domains, the T-DNA between the ATPase 1 To whom correspondence should be addressed: Stanford University, School domains, or both DNAs activate the ATPase reaction. Two of Medicine, Beckman Center B400, Dept. of Biochemistry, 279 Campus Dr., Stanford, CA 94305. Tel.: 650-723-9442; Fax: 650-723-6783; E-mail: lines of evidence suggest a role for the T-DNA in ATPase acti- [email protected]. vation. First, mutation of a residue lining the putative T-DNA AUGUST 18, 2006•VOLUME 281•NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 23395 Interdomain Communication in Topoisomerase II Oligo68bottom), the following duplexes were formed: a 40-bp duplex, a fluorescently labeled 40-bp duplex of the same sequence, a 46-bp duplex, and a 68-bp duplex. The duplexes are referred to throughout by their duplex length. The 68- and 40-bp-long DNA duplexes were derived from bp 63–130 and 87–126 in pBR322, respectively, and contain a known topo- isomerase II cleavage site (19). The 46-bp duplex was selected because it was efficiently cleaved by the enzyme (see supple- mental information). Before annealing, all oligodeoxynucleotides were purified by FIGURE 1. Proposed mechanism of topoisomerase II catalyzed DNA trans- denaturing PAGE except for the sequence Oligo40top-ROX, port (2, 3). G, G-DNA; T, T-DNA; E, topoisomerase II (ATPase domains are which was purchased high pressure liquid chromatography- colored light gray). Steps 1–4 are described in the Introduction. purified. As necessary, DNA duplexes were 5Ј-end-labeled with [␥-32P]ATP (MP Biomedicals) and T4-polynucleotide kinase site of the related enzyme Escherichia coli DNA gyrase abol- (New England Biolabs) according to the supplier’s instructions. ished the stimulation of ATP hydrolysis by DNA (14). Second, The 40-bp DNA duplex that carried the fluorescent dye 6- DNA stimulated ATP hydrolysis by truncated topoisomerase II carboxy-X-rhodamine (ROX),2 the 46-bp duplex, and 32P-la- Downloaded from lacking the G-DNA binding site (8, 15–17). Olland and Wang beled duplexes were further purified by nondenaturing PAGE. (16) have proposed, however, that this stimulation occurs The concentration of duplex DNA was determined by meas- because DNA assists in dimerizing the truncated subunits in a uring the absorption of the individual nucleotides after exhaus- manner that is not relevant to the normal reaction of the full- tive hydrolysis by Crotalus atrox phosphodiesterase I (Sigma) length enzyme and that the G-DNA is instead responsible for and DNase I (Roche Applied Science) (20). the DNA-dependent ATPase stimulation. The concentration of DNA duplex necessary to half-maxi- http://www.jbc.org/ One of the basic challenges to dissecting communication mally stimulate the ATPase activity at enzyme concentrations within topoisomerase II is the presence of two DNA binding low enough such that no or minimal sigmoidal shape of the sites, which complicates interpretation of effects from DNA. To DNA stimulation curve is observed ([E] Ͻ 50 nM; see Fig. 2 and duplex dissect the effects of G-DNA and T-DNA binding on the supplemental Fig. S4) is referred to as KM herein. The value duplex at Stanford University on February 12, 2016 ATPase and DNA cleavage activity in intact, full-length topoi- of KM was lower for the 68-bp duplex than the 46-bp and 68 bp ϭ ␮ 46 bp ϭ ␮ 40 bp ϭ somerase II, the occupancy of the sites needs to be controlled. 40-bp duplex (KM 0.15 M, KM 2.7 M, and KM 4 Plasmid DNA and long DNA duplexes can bind at one site or ␮M with saturating ATP; Fig. 2, supplemental Fig. S4, and data duplex bend to allow binding of a single DNA at both sites. To ensure not shown). The low KM of the 68-bp duplex allowed the use that a single DNA contacts only one binding site, we have used of saturating concentrations of enzyme with respect to DNA in short DNA duplexes. Binding of these duplexes was probed by the ATPase assay.
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