DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg2þ- mediated dynamic bending of gate-DNA Sanghwa Leea,b,1, Seung-Ryoung Junga,b,1, Kang Heoa,b, Jo Ann W. Bylc, Joseph E. Deweesec,d, Neil Osheroffc,e,2, and Sungchul Hohnga,f,b,2 aDepartment of Physics and Astronomy, fBiophysics and Chemical Biology, and bNational Center for Creative Research Initiatives, Seoul National University, Seoul 151-747, Korea; cDepartments of Biochemistry and eMedicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, TN 37232-0146; and dDepartment of Pharmaceutical Sciences, Lipscomb University College of Pharmacy, Nashville, TN 37204-3951 Edited by Martin Gellert, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, and approved December 30, 2011 (received for review September 27, 2011) Topoisomerase II resolves intrinsic topological problems of double- can damage chromosomal integrity. In fact, widely prescribed an- stranded DNA. As part of its essential cellular functions, the en- ticancer and antibacterial drugs initiate cell death by increasing zyme generates DNA breaks, but the regulation of this potentially the cellular concentration of topoisomerase II-mediated DNA dangerous process is not well understood. Here we report single- strand breaks (3, 7–9). Thus, the DNA cleavage reaction of type molecule fluorescence experiments that reveal a previously unchar- II topoisomerases needs to be tightly regulated. acterized sequence of events during DNA cleavage by topoisome- Despite the paramount importance of the cleavage reaction of rase II: nonspecific DNA binding, sequence-specific DNA bending, type II topoisomerases in biology and practical medicine, many and stochastic cleavage of DNA. We have identified unexpected fundamental questions concerning the regulation of the cleavage structural roles of Mg2þ ions coordinated in the TOPRIM (topoi- reaction remain unanswered. For instance, decades of biochem- somerase-primase) domain in inducing cleavage-competent DNA ical studies revealed that the DNA cleavage reaction occurs bending. A break at one scissile bond dramatically stabilized DNA only at specific sequences (13), but it is not understood how the BIOPHYSICS AND bending, explaining how two scission events in opposing strands cleavage sites are selected. In most in vitro assays, the cleavage can be coordinated to achieve a high probability of double-stranded efficiency of type II topoisomerases is very low (3). However, a COMPUTATIONAL BIOLOGY cleavage. Clamping of the protein N-gate greatly enhanced the rate mechanism to increase the enzymatic cleavage efficiency of type II and degree of DNA bending, resulting in a significant stimulation of topoisomerases has not yet been identified. In contrast to the low the DNA cleavage and opening reactions. Our data strongly suggest cleavage efficiency of type II topoisomerases, the probability of a that the accurate cleavage of DNA by topoisomerase II is regulated double-stranded break is extremely high, suggesting that cleavage through a tight coordination with DNA bending. of the two separate strands is efficiently communicated. However, the communication mechanism has not yet been delineated. N-gate clamping ∣ single-molecule FRET ∣ type II topoisomerase ∣ Here we report single-molecule fluorescence resonance energy G-segment selection ∣ indirect readout transfer (FRET) assays (14) that monitor the individual reaction steps of the interaction between human topoisomerase IIα (hTo- he double helical nature of DNA imposes intrinsic topological poIIα) and DNA. Similar methodologies using different labeling Tproblems during replication, repair, and transcription (1–3). schemes recently have been used to monitor opening of the Additionally, the topological state of the genetic material needs G-segment DNA and narrowing of the N-gate in Drosophila to- to be tightly regulated in order to promote proper biochemical poisomerase II and Escherichia coli gyrase, respectively (15–17). interactions between DNA and a variety of proteins (1–4). Topoi- Results of the present study strongly suggest the following: (i)a somerases are enzymes that resolve topological problems within sharp bend in DNA is dynamically induced by the enzyme as a the double helix by repeated cycles of DNA cleavage and ligation prerequisite to the cleavage reaction; (ii) although the DNA bind- (1–3, 5, 6). ing step appears to be nonspecific with regard to DNA sequence, As a subclass of the topoisomerase family, type II topoisome- bending was observed only with cleavable sequences, indicating rases are found in all organisms from bacteria to human, and even that the deformability of DNA sequences is an important deter- in some viruses (1–3, 5, 6). The essential roles of type II topoi- minant of G-segment selection. (iii) the interaction of divalent somerases in cell metabolism, differences between bacterial and metal ions with the highly conserved acidic residues in the human homologues, and hyperactivation of these enzymes in TOPRIM (topoisomerase-primase) domain is critical for DNA cancer cells have been utilized for clinical treatments of bacterial bending, revealing an unexpected structual role for divalent ions infections and numerous cancers (3, 7–9). in the enzymatic function of type II topoisomerases; (iv) a break Extensive studies for more than twenty years have established at one scissile bond dramatically increases the bending lifetime, that type II topoisomerases use a “two gate” mechanism for DNA providing a physical mechanism for the coordination of double- strand passage (3, 10, 11), in which a DNA duplex (the transport or T-segment) is transported through an enzyme-mediated tran- Author contributions: S.L., S.-R.J., N.O., and S.H. designed research; S.L., S.-R.J., and K.H. sient opening in a separate DNA duplex (the gate or G-segment). performed research; J.A.W.B., J.E.D., N.O., and S.H. contributed new reagents/analytic The directionality of strand passage is the N-terminal gate of the tools; S.L., S.-R.J., and K.H. analyzed data; and S.L., S.-R.J., N.O., and S.H. wrote the paper. enzyme to the C-terminal gate. As a result of the double-stranded The authors declare no conflict of interest. DNA passage mechanism, each catalytic event changes the linking This article is a PNAS Direct Submission. number of DNA by two. The transport of the T-segment through Freely available online through the PNAS open access option. G N the -segment is thought to be initiated by the -gate clamping 1S.L. and S.-R.J. contributed equally to this work. motion induced by the binding of ATP to the enzyme (3, 6, 10, 12). 2To whom correspondence may be addressed. E-mail: [email protected] or Although the double-stranded DNA breaks generated by type [email protected]. II topoisomerases are essential for the cellular functions of these This article contains supporting information online at www.pnas.org/lookup/suppl/ enzymes, it is a dangerous process in which an aberrant operation doi:10.1073/pnas.1115704109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1115704109 PNAS ∣ February 21, 2012 ∣ vol. 109 ∣ no. 8 ∣ 2925–2930 Downloaded by guest on September 24, 2021 stranded breaks; (v) the rate and degree of DNA bending are dissociation events of the enzyme (Fig. 1C). In the presence of greatly enhanced by clamping of the protein N-gate, resulting 5mMMg2þ, however, the situation changed dramatically. Large in a great stimulation of DNA cleavage and opening, which is FRET jumps (from E ¼ 0.27 to E ¼ 0.50) were observed in some disfavored under normal conditions. Overall, our work provides of the enzyme-DNA binding events (Fig. 2A, top), indicating that evidence that Mg2þ-mediated DNA bending by topoisomerase II a substantial amount of DNA deformation was stochastically in- is the basis for both the cleavage site selection and the regulation duced by hTopoIIα. Similar FRET jumps were observed in the of double-stranded DNA cleavage by the enzyme. presence Ca2þ (SI Appendix, Fig. S6). The FRET jump, however, was not observed when a noncleavable DNA sequence was used Results as the substrate (24) (non-clv in SI Appendix, Fig. S1; Fig. 2A, Observation of Single-Enzyme Binding Events. A partial DNA duplex middle), but became more frequent and stable when a nick containing a central cleavage site for hTopoIIα (18) and flanking was introduced in one of the two scissile bonds of the cleavage FRET probes (Cy3 and Cy5) was prepared [clv (DNA construct) sequence (clv-nick in SI Appendix, Fig. S1; Fig. 2A, bottom; SI in SI Appendix, Fig. S1; Fig. 1A]. The duplex has a biotinylated Appendix, Fig. S7B). Introduction of a nick in non-clv, however, single-stranded overhang to avoid any potential steric hindrance did not induce any FRET jump during enzyme binding events (SI caused by surface immobilization. After immobilizing DNA Appendix, Fig. S8). Combining the above observation with the molecules on a polymer-coated quartz surface, hTopoIIα was knowledge that a nick in one strand greatly increases the cleavage delivered into a detection chamber, and the fluorescence signals efficiency of the opposite strand (25, 26), we conclude that DNA of single DNA molecules were monitored using a total-internal- deformation induced by hTopoIIα occurs in a sequence-specific reflection fluorescence microscope (Fig. 1B). fashion with a strong correlation between the cleavage efficiency In the absence of enzyme, fluorescence intensities showed a and the population of the deformed state (Fig. 2A, right). single-state behavior with small fluctuations limited by shot-noise Sharp bending of the G-segment DNA is supported by a num- (SI Appendix, Fig. S2). Upon addition of hTopoIIα, however, ber of experiments including recent high-resolution X-ray struc- large intensity jumps with appreciable dwell times were observed tures, atomic force microscopy, and FRET (27–31). With this (Fig. 1C). The frequency of the intensity jumps increased linearly context in mind, the FRET jumps specifically observed only in with enzyme concentration (Fig.