Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA Zheng Yang, James F. Carey and James J. Champoux Department of Microbiology, School of Medicine, University of Washington, Seattle, WA, USA Introduction Type I DNA topoisomerases relax supercoils by intro- ducing a transient single-strand break in the DNA. These enzymes are classified into type IA and type IB subfamilies based on the polarity of attachment to the cleaved DNA [1–3]. The members of the two subfami- lies share no sequence homology and are further dis- tinguished by their substrate requirements and mechanisms of relaxation. Type IA subfamily members require a single-stranded region to bind DNA, become attached to the 5¢ end upon cleavage, and only relax negatively supercoiled DNA in the presence of divalent cations such as Mg 2+ . Escherichia coli DNA topo- isomerase I is the prototype of the type IA subfamily. Type IB subfamily members bind double-stranded DNA, become attached to the 3¢ end of the cleaved strand, and relax both positive and negative supercoils. ATP or divalent cations are not required for the type IB enzymes, although Mg 2+ and Ca 2+ enhance the rate of relaxation [4]. The cleavage–religation reaction catalyzed by human DNA topoisomerase I, the prototypical type IB enzyme, is essential for many biological processes, Keywords competition binding assay; DNA topology; node binding; supercoiled DNA; topoisomerase I Correspondence J. J. Champoux, Department of Microbiology, University of Washington, Box 357242, Seattle, WA 98195-7242, USA Fax: +1 206 543 8297 Tel: +1 206 543 8574 E-mail: champoux@u.washington.edu (Received 7 July 2009, revised 9 August 2009, accepted 11 August 2009) doi:10.1111/j.1742-4658.2009.07270.x Human topoisomerase I binds DNA in a topology-dependent fashion with a strong preference for supercoiled DNAs of either sign over relaxed circu- lar DNA. One hypothesis to account for this preference is that a second DNA-binding site exists on the enzyme that mediates an association with the nodes present in supercoiled DNA. The failure of the enzyme to dimer- ize, even in the presence of DNA, appears to rule out the hypothesis that two binding sites are generated by dimerization of the protein. A series of mutant protein constructs was generated to test the hypotheses that the homeodomain-like core subdomain II (residues 233–319) provides a second DNA-binding site, or that the linker or basic residues in core subdo- main III are involved in the preferential binding to supercoiled DNAs. When putative DNA contact points within core subdomain II were altered or the domain was removed altogether, there was no effect on the ability of the enzyme to recognize supercoiled DNA, as measured by both a gel shift assay and a competition binding assay. However, the preference for supercoils was noticeably reduced for a form of the enzyme lacking the coiled-coil linker region or when pairs of lysines were changed to glutamic acids in core subdomain III. The results obtained implicate the linker and solvent-exposed basic residues in core subdomain III in the preferential binding of human topoisomerase I to supercoiled DNA. Abbreviations Dcap, NH 2 -terminal truncation of human topoisomerase beginning at residue 433; GST, glutathione S-transferase; topo31, a fragment of human topoisomerase I extending from residues 175–433; topo56, COOH-terminal truncation of topo70 missing the last 126 amino acids; topo58, COOH-terminal truncation of topo70 missing the last 106 amino acids; topo70, NH 2 -terminal truncation of human topoisomerase I missing the first 174 amino acids; topo70DL, a form of topo70 missing linker residues 660–688. 5906 FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS including DNA replication, transcription and recombi- nation [2,3]. Strand cleavage is initiated by nucleophilic attack of the O4 atom of the active site tyrosine on the scissile phosphate in the DNA, resulting in the cova- lent attachment of the enzyme to the 3¢ end of the broken strand [2]. Rotation of the duplex region downstream of the break site relieves any supercoiling strain in the DNA prior to religation and release of the topoisomerase [5,6]. Human DNA topoisomerase I is composed of 765 amino acids and has a molecular mass of 91 kDa. On the basis of sequence comparisons, limited proteolysis studies and the crystal structure of the enzyme [7,8], four domains have been identified in the protein: an NH 2 -terminal domain (Met1-Gly214), a core domain (Ile215-Ala635), a linker domain (Pro636-Lys712) and a COOH-terminal domain (Gln713-Phe765) (Fig. 1). The NH 2 -terminal domain is unstructured, poorly con- served, highly charged and dispensable for the DNA relaxation activity in vitro . It contains nuclear localiza- tion signals and was shown to interact with nucleolin, the SV40 large T antigen, p53, and possibly certain transcription factors [9–13]. Topo70 is a truncated form of human topoisomerase I that lacks residues 1–174 of the NH 2 -terminal domain, yet retains full enzymatic activity [7] and a preference for binding su- percoiled DNA. The core domain is highly conserved and more protease-resistant than the other domains. The poorly-conserved linker domain is highly charged and forms an anti-parallel coiled-coil structure that connects the core domain to the COOH-terminal domain. The linker protrudes from the body of the protein and, instead of tracking with the axis of a bound DNA helix, angles away from DNA. The COOH-terminal domain is highly conserved and con- tains the active site tyrosine, Tyr723. When separately expressed, the COOH-terminal and core domains can associate in vitro to reconstitute wild-type levels of enzymatic activity, demonstrating that the linker domain is not required for activity [4,7,14]. The co-crystal structure of human topoisomerase I with bound DNA indicates that the core domain can be further divided into three subdomains [8] (Fig. 1). Core subdomain I (residues 215–232, 320–433) and core subdomain II (residues 233–319) form the Cap structure of the enzyme and cover one side of the DNA. Core subdomain III (residues 434–635) contains all the residues implicated in catalysis except Tyr723 and cradles the DNA on the side opposite of the Cap [5,8]. Although there is little sequence similarity, the fold of the subdomain II is very similar to that of a homeodomain found in a family of DNA-binding proteins. For example, residues 244–314 of the core subdomain II superimpose on the POU homeodomain of the Oct-1 transcription factor with an rmsd of only 3.0 A ˚ [8,15]. This observation suggests that core sub- domain II, which forms part of the exposed Cap, could represent a second DNA-binding site distinct from the substrate binding channel observed in the co-crystal structure. However, the conserved residues that are involved in base-specific contacts in the POU homeodomain are absent in core subdomain II of human topoisomerase I, suggesting that, if sub- domain II interacts with DNA, it does so with low affinity and likely without sequence specificity [15,16]. It has been proposed that topoisomerases relax the negative and positive supercoils generated by the trans- location of an RNA polymerase along the DNA during transcription [17]. In support of this model, eukaryotic type IB topoisomerases have been found to associate with transcriptionally active genes and have been reported to interact directly with the transcription machinery [18–26]. Eukaryotic type IB topoisomerases have also been shown to provide the swivels for the Fig. 1. Crystal structure of human topoisomerase I. Core subdo- mains I, II and III are colored yellow, blue and red, respectively, with the linker and C-terminal domains colored orange and green, respectively. The Cap and Linkers regions are labeled along with the amino acid residues that were changed in the present study. Amino acids in core subdomain II (His266, Lys299 and Ser306) that were changed to glutamic acid in the combinations indicated in the text are shown in ball and stick and colored magenta. The three amino acids in the linker (Lys650 ⁄ Lys654 ⁄ Gln657) that were simultaneously changed to alanine are similarly depicted and colored brown. The four amino acids in the linker (Lys679 ⁄ Lys682 ⁄ Lys687 ⁄ Lys689) that were simultaneously changed to serine are colored gray. Surface-exposed lysine residues in core subdomain III (Lys466 ⁄ Lys468 and Lys545 ⁄ Lys549) that were pairwise mutated to glutamic acid are colored black. Z. Yang et al. Supercoil binding by topoisomerase I FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS 5907 relaxation of positive supercoils during DNA replica- tion [20,27–31]. The mechanism for recruiting DNA topoisomerase I to transcriptionally active and repli- cating DNA remains unclear, although several studies have shown that the enzyme prefers to bind super- coiled over relaxed DNA [32–37]. Because the enzyme binds supercoiled DNA irrespective of the sign of the supercoils, Zechiedrich and Osheroff [36] hypothesized that topoisomerase I specifically binds at a node where two duplex regions of the supercoiled DNA cross and also provided electron microscopic evidence in support of this hypothesis [36]. The structural basis for the preferential binding of human topoisomerase I to supercoiled DNA is unknown but, if node recognition is important, then it is likely that the binding involves an interaction with two regions of DNA at the point of crossing. One hypothesis to explain how the enzyme provides two DNA-binding sites to stabilize an interaction with a DNA node is to assume that it binds as a dimer (Fig. 2A). An alternative hypothesis is that, in addition to the substrate binding channel identified in the crys- tal structure of the protein (Fig. 1) [8], there is a second DNA-binding site present on the protein that stabilizes an interaction at a DNA node (Fig. 2B). In the present study, we performed experiments designed to distinguish between these possible explanations for the preference of topoisomerase I for supercoils. Results Human topoisomerase I does not dimerize in the absence or presence of DNA We previously used a gel filtration assay to demon- strate that, although topo70DL, a mutant form of topo70 missing a portion of the linker (i.e. linker residues 660–688), formed dimers through a domain swapping mechanism, no dimerization of WT topo70 was detectable under the same conditions [4,38]. Because these earlier experiments were carried out in the absence of DNA, we wanted to test whether dimers could form in the presence of DNA. In the present study, we used a glutathione S-transferase (GST) pull-down assay to determine whether topo70 that was already covalently bound to a DNA oligonu- cleotide could dimerize. GST-topo70 was incubated with free topo70 in the absence or presence of an oli- gonucleotide suicide substrate, and any protein bound to GST-topo70 was collected by adsorption to gluta- thione S-Sepharose beads and analyzed by SDS– PAGE. Control experiments showed that free topo70 did not bind to either GST alone or to the beads (Fig. 3, lanes 6 and 7). Under the same conditions, no topo70 was found associated with the bead-bound GST-topo70 either in the absence or presence of DNA (Fig. 3, lanes 2 and 3, respectively). The slower migrat- ing species of the doublet observed in lane 3 in Fig. 3 is the result of suicide cleavage and shows that approx- imately half of the GST-topo70 contained covalently bound oligonucleotide DNA. Thus, these results con- firm our earlier finding that topo70 does not dimerize when free in solution and also extend the results to show that, even when bound to DNA after suicide cleavage, dimerization does not occur. Fig. 2. Alternative modes for topoisomerase I binding to a DNA node. (A) Node binding occurs through dimerization of topoisomer- ase I. (B) Node binding is mediated by two DNA-binding sites on a single molecule of topoisomerase I. Fig. 3. GST pull-down experiment to test for dimerization. The indi- cated combinations of GST-topo70, topo70 and GST were incu- bated with and without a suicide DNA oligonucleotide and mixed with glutathione Sepharose 4B beads (GSH beads). The beads were collected by centrifugation, washed and the samples were analyzed by SDS-PAGE. Lane 1, protein markers with sizes (kDa) indicated along the left side of the gel. Lanes 4 and 5 contain GST- topo70 and topo70 size markers, respectively. The GST protein in lane 6 was run off the gel in this analysis. Although all of the samples were analyzed on the same gel, lanes with unrelated data were removed digitally at the places indicated by the thin vertical lines. Supercoil binding by topoisomerase I Z. Yang et al. 5908 FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS DNA-binding properties of mutant proteins as measured by a gel shift assay A structural alignment of core subdomain II of human topoisomerase I with the POU homeodomain of Oct-1 indicated that the residues making base specific contacts with the DNA in the homeodomain are not conserved in the core subdomain II. However, basic residues K25, R46 and R53 of the POU homeodomain that make hydrogen bonds with phosphates in the bound DNA correspond to residues His266, Lys299 and Ser306 in core subdomain II of human topo- isomerase I (Fig. 1). All three of these residues are conserved among known eukaryotic topoisomerase I sequences. To test whether these amino acids mediate an interaction with DNA that accounts for node bind- ing by the enzyme, site-directed mutagenesis was used to replace these residues with glutamic acid in topo70. These changes would be predicted to disrupt an inter- action with the DNA phosphate backbone, but have a minimal effect on the overall enzyme structure because all three are in a solvent-exposed region. Because the assays to detect the preferential binding to supercoiled DNA require a catalytically inactive form of the pro- tein [37], a mutation in the active site tyrosine (Y723F) was also introduced into the proteins. Topo70 capKS- E ⁄ Y723F and topo70 capHKS-E ⁄ Y723F were expressed and purified from recombinant baculovirus- infected insect SF-9 cells. The following proteins were similarly purified for use in these assays: a reconsti- tuted form of the protein lacking the linker, compris- ing a COOH-terminal truncation of topo70 missing the last 126 amino acids (topo56) plus the Y723F mutant form of the COOH-terminal domain (topo6.3), the catalytically inactive NH 2 -terminal truncation of human topoisomerase beginning at residue 433 (Dcap) [39] and a fragment of human topoisomerase I extend- ing from residues 175–433 (topo31) (Fig. 4). The various forms of the topoisomerase protein described above were mixed with an equimolar mixture of supercoiled, nicked circular and linear pBluescript KSII(+) DNAs, and a gel shift assay [40–44] was used to analyze the preference of the proteins for the different topological forms of DNA. For the positive Fig. 4. Human topoisomerase I fragments used in the DNA-binding studies. (A) The four domains of full-length human topoisom- erase I (topo I) are shown above the various constructs used in the binding studies: topo70, a 70 kDa NH 2 -terminally truncated protein that starts with an engineered Met upstream of Lys175; topo58, a COOH-termi- nal deletion of topo70, ending at Ala659; topo31, a COOH-terminal deletion of topo70 ending at Ser433; Dcap, an NH 2 -terminal truncation starting at Ser433; topo56 ⁄ 6.3, a reconstituted protein comprising the core domain from Lys175 to Thr639 and the COOH-terminal domain from Lys713 to the COOH terminus (Phe765). (B) SDS-PAGE analysis of 2 lg of the indicated purified pro- teins. Lane 1, protein markers with sizes (kDa) indicated along left side of the panel; lane 2, topo70 Y723F; lane 3, topo70 capKS- E ⁄ Y723F; lane 4, topo70 capHKS-E ⁄ Y723F; lane 5, topo56 ⁄ 6.3 Y723F (6.3 kDa fragment of topo6.3 Y723F was run off the bottom of the gel); lane 6, Dcap; lane 7, topo31; lane 8, protein markers; lane 9, topo70 K466- 468E ⁄ Y723F; lane 10, topo70 K545- 549E ⁄ Y723F. Z. Yang et al. Supercoil binding by topoisomerase I FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS 5909 control protein, topo70 Y723F, the mobility of the supercoiled DNA was reduced, with essentially no effect on the mobility of either the nicked or linear DNAs at the two lowest protein concentrations (Fig. 5A, com- pare lanes 2 and 3 with lane 1). As the amount of topo70 Y723F protein was increased, the supercoiled DNA was shifted further and, to a lesser extent, both the linear and nicked DNA bands became shifted as well (Fig. 5A, lanes 4 and 5). These results confirmed the earlier find- ing that topo70 Y723F has a preference for supercoiled over linear and nicked DNA [37]. Topo31, which corre- sponds to the Cap region of human topoisomerase I, provides a convenient nonspecific negative control for this analysis. As shown in Fig. 5A, lanes 22–26, all three forms of the plasmid DNA responded equally to increasing concentrations of the topo31 fragment, con- sistent with a lack of preference for one form over another. A higher concentration of topo31 was required to effect a gel shift, reflecting the lower affinity of the protein for DNA compared to topo70. Both topo70 capKS-E ⁄ Y723F and topo70 capHKS- E ⁄ Y723F retained the preference for binding super- coiled DNA (Fig. 5A, lanes 7–10 and 12–15), ruling out Cap residues His266, Lys299 and Ser306 as con- tributors to the preferential binding to supercoils. To further test the possible involvement of the core subdo- main II in the preferential binding to supercoiled DNA, the Dcap mutant lacking core subdomains I and II was also tested in the gel shift analysis (Fig. 5A, lanes 17–20). Dcap contains core subdomain III, the linker domain, and the COOH-terminal domain (resi- dues 433–765) (Fig. 4A), and is catalytically inactive, despite containing all of the residues known to be directly involved in catalysis [39]. At the lower concen- trations of the Dcap protein, the supercoiled DNA was selectively shifted upon binding, although the magni- tude of the shift was less compared to that observed with the topo70 protein (Fig. 5A, compare lanes 17–20 with lanes 1–5). This reduction in the shift most likely resulted from the two-fold lower affinity of the Dcap for DNA [39] and the lower molecular weight of Dcap (41 kDa) compared to topo70 (71 kDa). Thus, deletion of the Cap region that includes subdomain II did not eliminate the preference for supercoiled DNA, indicat- ing that core subdomain II is dispensable for the preferential binding of topoisomerase I to supercoils. Although the band corresponding to the supercoiled DNA was selectively shifted in the presence of topo70 Y723F and all of the mutant proteins except topo31, we wanted to formally rule out the possibility that the Fig. 5. DNA-binding measured by an agarose gel shift assay. (A) Two-fold serial dilutions of the indicated proteins were incubated with equal amounts of supercoiled, linear and nicked pBluescript KSII(+) plasmid DNA and analyzed by electrophoresis in an agarose gel as described in the Experimental procedures. The mobilities of unshifted supercoiled, linear and nicked DNAs are indicated along the right side. Lanes 1, 6, 11, 16, 21 and 27 contain DNA alone; lanes 2–5 contain 0.88, 1.75, 3.5 and 7 pmol of topo70 Y723F, respectively; lanes 7–10 contain 0.88, 1.75, 3.5 and 7 pmol of topo70 capKS-E ⁄ Y723F, respectively; lanes 12–15 contain 0.88, 1.75, 3.5 and 7 pmol of topo70 capHKS-E ⁄ Y723F, respectively; lanes 17–20 contain 0.88, 1.75, 3.5 and 7 pmol of Dcap, respectively; and lanes 22-26 contain 0.88, 1.75, 3.5, 7 and 14 pmol of topo31, respectively. The white spaces demarcate separate gel analyses. (B) Same experimental design as in (A) for the indicated proteins. Lanes 1, 6 and 11 are DNA alone; lanes 2–5 contain 0.88, 1.75, 3.5 and 7 pmol of topo70 Y723F, respectively; and lanes 7–10 contain 0.88, 1.75, 3.5 and 7 pmol of topo56 ⁄ 6.3 Y723F, respectively. Supercoil binding by topoisomerase I Z. Yang et al. 5910 FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS proteins bound to the supercoiled, linear and nicked DNAs equally well, but only the supercoiled DNA shift was detected visually because of its greater initial mobility. Therefore, the gel shift assay was repeated using topo70 Y723F or topo70 capHKS-E ⁄ Y723F that had been previously labeled with 32 P using protein kinase C. The autoradiograph of the agarose gel showed that the majority of the labeled proteins were associated with the shifted supercoiled DNA and that the amount of bound label correlated with the extent of the shift (Fig. 6, lanes 2, 3, 5 and 6). Furthermore, label was only associated with the nicked and linear DNAs at the protein concentration where a mobility shift of these species was also detected (Fig. 6, lanes 3 and 6). These results validated the gel shift assay and confirmed that the selective shift of the supercoiled DNA band results from preferential binding. To further define the region that is involved in the preferential binding to supercoiled DNA, we repeated the assays using a form of human topoisomerase I reconstituted from a mixture of topo56 and topo6.3 Y723F (Fig. 4A). This reconstituted protein contains only the core and COOH-terminal domains and com- pletely lacks the linker region (Fig. 1). When tested in the gel shift assay, topo56 ⁄ 6.3 Y723F retained a pref- erence for supercoiled DNA, although the preference was reduced compared to that of the topo70 Y723F (Fig. 5B). For example, although only the supercoiled DNA was shifted by both topo70 Y723F and topo56 ⁄ 6.3 Y723F at the lowest protein concentration tested (Fig. 5B, lanes 2 and 7), at the higher protein concentrations where mainly the supercoiled DNA was shifted by topo70 Y723F, the reconstituted enzyme shifted the linear and nicked DNAs as well (Fig. 5B, in particular, compare lane 4 with lane 9). These results suggest that an intact linker region is necessary for the full manifestation of the preference for super- coiled DNA but, in its absence, the enzyme can still distinguish to a limited extent a supercoiled from a nonsupercoiled DNA. Competition binding assays To verify these results by an independent method and to provide a more quantitative measure for the binding of the various proteins to supercoiled DNA, we employed a filter binding assay similar to the one we used previously [37]. Unlabeled nicked and supercoiled SV40 DNAs were used separately as competitors for the binding of 3 H-labeled nicked SV40 DNA to cata- lytically inactive (Y723F) mutant forms of topo70. The competition assays were carried out for topo70 cap- HKS-E ⁄ Y723F and 4cap and the results were com- pared with those obtained for topo70 ⁄ Y723F. For all three proteins, the competition profile for the like com- petitor (nicked DNA) exhibited a half-maximum at the expected 1 : 1 ratio of competitor to labeled DNA (Fig. 7A, closed symbols), whereas only approximately one-tenth as much supercoiled competitor was required to reduce the binding of the labeled nicked DNA to the 50% level (Fig. 7A, open symbols). The competi- tion profile of topo56 ⁄ 6.3 Y723F for the supercoiled DNA showed that the amount of supercoiled DNA needed to compete to the 50% level was approximately one-third as much as for the nicked DNA (Fig. 7B). These results are consistent with the gel shift assays and confirm that topo70 Y723F, topo70 capHKS- E ⁄ Y723F and Dcap have a strong preference for supercoiled DNA over nicked DNA, whereas the reconstituted topo56 ⁄ 6.3 Y723F lacking the linker has a reduced ability to discriminate supercoiled from nicked DNA. Because the above results implicate the linker in the preference for binding supercoiled DNA, we wanted to investigate whether the clusters of positively-charged amino acids in the linker region are required for this effect. To test this possibility, we generated two mutant forms of topo70 Y723F, each of which elimi- nates the positive charges associated with clusters of basic amino acids within one of the a-helices of the lin- ker region (a18). The changes in one of the mutant proteins were K650A ⁄ K654A ⁄ Q657A and in the Fig. 6. Gel shift assay with 32 P labeled proteins. (A) Agarose gel shift assay as described for Fig. 5 using 32 P labeled topo70 Y723F and topo70 capHKS-E ⁄ Y723F. Lanes 1 and 4, DNA alone; lanes 2 and 3 contain 1.75 and 3.5 pmol of topo70 Y723F, respectively; lanes 5 and 6 contain 1.75 and 3.5 pmol of topo70 capHKS- E ⁄ Y723F, respectively. (B) Autoradiogram of the gel shown in (A). The mobilities of unshifted supercoiled, linear and nicked DNAs are indicated along the right side. Z. Yang et al. Supercoil binding by topoisomerase I FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS 5911 second were K679S ⁄ K682S ⁄ K687S ⁄ K689S (Fig. 1). These proteins are referred to as topo70 linkerKKQ- A ⁄ Y723F and topo70 linker4K-S ⁄ Y723F, respectively. When these proteins were used in the competition binding assay, the ratio of unlabeled supercoiled com- petitor to labeled nicked DNA that was required for half-maximal binding was offset from the ratio for the nicked or like competitor by the same amount for the mutants as for the topo70 Y723F protein (Fig. 8A). The magnitude of this offset was slightly less for the competition profiles in Fig. 8A compared to that observed in Fig. 7A because the preparation of unla- beled supercoiled competitor used in this experiment contained a slightly higher percentage of nicked mole- cules ( 20% compared with the previous  5%, data not shown). On the basis of these results, we conclude that the absence of either of these two clusters of basic amino acid within the linker does not affect the ability of the protein to preferentially bind supercoiled DNA. The solvent-exposed region of the core subdo- main III distal from the Cap represents yet another Fig. 8. (A) Filter binding assays comparing unlabeled supercoiled and nicked SV40 DNAs as competitors for 3 H-labeled nicked SV40 DNA-binding to topoisomerase variants containing multiple amino acid changes in the linker domain: topo70 Y723F (nicked competi- tor, solid squares; supercoiled competitor, open squares); topo70 linker4K-S ⁄ Y723F (nicked competitor, solid triangles; supercoiled competitor, open triangles); and topo70 linkerKKQ-A ⁄ Y723F (nicked competitor, solid diamonds; supercoiled competitor, open diamonds, dashed line). (B) Filter binding assays for topoisomerase variants containing mutations at exposed lysine residues in the core domain of the enzyme: topo70 Y723F (nicked competitor, solid diamonds, supercoiled competitor, open diamonds); topo70 K466-468E Y723F (nicked competitor, solid squares, supercoiled competitor, open squares); and topo70 K545-549E Y723F (nicked competitor, solid tri- angles, supercoiled competitor, open triangles). For topo70 Y723F, the values plotted are the mean of seven independent determina- tions and, for the two mutant proteins, the values are the mean of six independent determinations. Fig. 7. Filter binding assays comparing unlabeled supercoiled and nicked SV40 DNAs as competitors for 3 H-labeled nicked SV40 DNA-binding to topoisomerase I constructs. (A) The results of the competition assay for topo70 Y723F (nicked competitor, solid squares; supercoiled competitor, open squares), topo70 capHKS- E ⁄ Y723F (nicked competitor, solid triangles; supercoiled competi- tor, open triangles) and Dcap (nicked competitor, solid diamonds; supercoiled competitor, open diamonds). (B) Results for the compe- tition assay for topo56 ⁄ 6.3 Y723F (nicked competitor, solid circles; supercoiled competitor, open circles). Supercoil binding by topoisomerase I Z. Yang et al. 5912 FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS region of the protein that might provide a binding interface for a second DNA-binding site. To examine this possibility, we generated mutant proteins in which pairs of positively-charged lysine residues within core subdomain III were changed to glutamates (Fig. 1) and tested these proteins in the competition binding assay. As shown in Fig. 8B, the competition profiles of the nicked competitor DNA for the topo70 K466- 468E ⁄ Y723F and topo70 K545-549E ⁄ Y723F proteins are identical to the profile for the control topo70 Y723F protein (Fig. 8B, closed symbols) but, impor- tantly, the supercoiled DNA did not compete as well for the binding to the two mutant proteins as it did for the binding to the control topo70 Y723F protein (Fig. 8B, compare the open squares and triangles with the open diamonds). To be certain that these differ- ences were significant, multiple experiments were per- formed to determine the mean value for the ratio of unlabeled nicked to supercoiled competitor required to reduce binding to the 50% level. For the positive con- trol topo70 Y723F, this ratio (±SD) was found to be 8.6 ± 3.9 (seven repeats), which is consistent with the earlier determinations, whereas the corresponding ratios for topo70 K466-468E ⁄ Y723F and topo70 K545-549E ⁄ Y723F were 4.1 ± 1.1 and 4.6 ± 1.7, respectively (six repeats). Using the t-test, these differ- ences of the ratios for the two mutant proteins from the control are significant at P < 0.05, and thus the mutant proteins have a reduced ability to discriminate supercoiled from nonsupercoiled DNA. Discussion Although protein–protein interactions have been impli- cated in targeting topoisomerase I to supercoiled sub- strates in vivo [21,24–26], when given a choice of supercoiled and relaxed substrates in the absence of other proteins in vitro, the enzyme exhibits a prefer- ence for binding to the supercoiled DNA [32–37]. Because this intrinsic preference for supercoils is inde- pendent of the sign of the supercoiling [37,45], it is likely the DNA feature being recognized by the enzyme is a DNA node [36], a structural element that is shared by DNAs with positive and negative super- coils. In the absence of DNA, the topoisomerase I protein is a bi-lobed structure that exists in an open clamp conformation [5]. Upon binding DNA, the clamp closes around the duplex to form a clearly- defined channel that interacts with the DNA backbone over a length of approximately 6 bp (Fig. 1) [8]. The simplest model to explain node recognition by the enzyme assumes that, in addition to this well-charac- terized DNA-binding channel, the protein has a second DNA-binding region that stabilizes the interaction with a DNA crossing. Here, we consider four struc- ture-related hypotheses that could explain node bind- ing. First, the bent structure of a supercoiled duplex could be a feature that is recognized by a single topo- isomerase I protein without the need for a second DNA-binding site. Second, a topoisomerase I homodi- mer could provide two DNA-binding sites on the same protein molecule (Fig. 2A). Third, core subdomain II, which structurally resembles a homeodomain and is an exposed feature of the Cap (Fig. 1), could constitute a second DNA-binding site on the protein. Fourth, clusters of basic residues in core subdomain III, and the linker on the side of the protein distal from the Cap, could mediate DNA-binding at a node. For some proteins, the preference for binding to supercoiled DNA is related to the tendency of the proteins to cause DNA bending. For example, high- mobility group proteins [44,46–50] and the p53 protein [40–43,51] preferentially bind supercoiled DNA and, in both cases, it was shown that the proteins bend DNA. Moreover, in the case of the high-mobility group pro- teins, the DNA bending capacity correlates with the supercoiled DNA-binding [50]. In the crystal structure of the human topoisomerase I-DNA complex, the 22 bp DNA substrate does not show any bending deformation and is an almost perfect B-shaped helix [8]. This observation suggests that the preference of human topoisomerase I for supercoiled DNA is not the result of an attraction of the enzyme for bent DNA. In a previous study [38], we showed that the topo70DL form of human topoisomerase I missing part of the coiled-coil linker domain could form dimers through a domain swapping mechanism involving the core and COOH-terminal domains of the two subunits. We hypothesized that the shortened linker in the mutant enzyme destabilized the interaction between the COOH-terminal and core domains, enabling the COOH-terminal domain of one protein to occupy its binding site in the core domain of the other protein and vice versa. Consistent with this suggestion, we were unable to detect dimerization of free wild-type enzyme containing the normal length linker [4,38]. However, these results did not rule out the possibility that dimerization of the enzyme only occurs after the first molecule of enzyme is already bound to DNA. In this regard, it was shown that a molecule of topoisom- erase I that is covalently trapped on DNA after suicide cleavage recruits another molecule of enzyme to cleave approximately 13 bp upstream of the trapped enzyme [52]. Although the basis for dimerization in this case is unknown, this interaction between two enzyme Z. Yang et al. Supercoil binding by topoisomerase I FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS 5913 molecules is unlikely to mediate node binding because the second molecule of enzyme is bound to the DNA immediately adjacent to the one already trapped on the DNA. For our GST pull-down assay, we deliber- ately chose an oligonucleotide that was too short to permit this type of side-by-side contact (total duplex length 14 bp) to assay for DNA-mediated dimeriza- tion. Importantly, under these conditions, we show that a topoisomerase I molecule covalently bound to DNA after suicide cleavage does not bind another molecule of the enzyme. These results rule against the hypothesis that dimerization of topoisomerase I accounts for the preference of the enzyme for super- coiled DNA. In previous studies [36,37] demonstrating a prefer- ence of topoisomerase I for supercoils, the full length enzyme was used. In the present study, we demonstrate that topo70, a form of the enzyme missing residues 1–174 that constitute most of the N-terminal domain, also preferentially binds supercoiled over relaxed DNA. This observation rules out this portion of the N-terminus as a region of the enzyme that provides a second DNA-binding site involved in node recogni- tion. In the present study, we tested whether the homeo- domain-like region within the Cap of the enzyme (core subdomain II) constitutes a second DNA-binding site on the enzyme that mediates the preference for super- coils (Fig. 2B). Alignment of the sequences of human topoisomerase I and the Oct-1 homeodomain revealed three amino acids within core subdomain II of the Cap that might be expected to interact with the negatively- charged DNA backbone and form the basis for a sec- ond DNA-binding site on the enzyme (His266, Lys299 and Ser306) (Fig. 1). Replacing all three of these resi- dues with a glutamic acid residue or complete deletion of the Cap region (Dcap) had no effect on the ability of the resulting proteins to preferentially bind super- coiled DNA when assayed by either a gel shift assay or a competition binding assay. These results rule out the hypothesis that an interaction with a node is medi- ated by a second DNA-binding site localized to core subdomain II of the enzyme. The results obtained in the present study with respect to topo56 ⁄ 6.3 Y723F, a reconstituted enzyme completely missing the linker region, reveal that this form of the enzyme has a reduced preference for supercoiled DNA compared to the wild-type enzyme. In a study carried out prior to the availability of the co-crystal structure of topoisomerase I [8], we exam- ined the substrate binding preference of topo58, a form of the protein now known to contain the core domain and one third of the linker region (residues 175–659) (Fig. 4). At the time, we concluded that the binding properties of a COOH-terminal truncation of topo70 missing the last 106 amino acids (topo58) was similar to those of topo70 Y723F, but a re-examination of these older data [37] reveals that, similar to the recon- stituted topo56 ⁄ 6.3 Y723F investigated in the present study, topo58 alone exhibits a reduced preference for supercoiled DNA. Taken together, these observations suggest that an intact linker region of the enzyme is necessary for the full manifestation of the preference for supercoils. It is noteworthy that the elimination of either of the clusters of basic amino acids within the linker region (Fig. 1) does not affect the preference of the enzyme for supercoiled DNA. Our interpretation of this finding is that the contribution of the linker to node binding relates to how the linker influences local protein structure rather than via the formation of a second DNA-binding site that makes direct amino acid side chain contacts with the DNA backbone. In this regard, it is noteworthy that the linker region is not only remarkably flexible [53], but also mutations that affect its flexibility can influence the structure of the protein at distant sites [54]. Unlike the linker where the evidence rules out a direct interaction between basic amino acids and the DNA in node binding, mutational studies within core subdomain III indicate that reversing the charge on pairs of basic, surface-exposed amino acids (K466 ⁄ K468 and K545 ⁄ K549) (Fig. 1) has a significant impact on the preferential binding of the topoisomer- ase to supercoiled DNA. Notably, these lysine residues are conserved in the topoisomerase I protein in most higher eukaryotes. (Fig. 9). These results suggest that basic amino acids within core subdomain III contrib- ute to node binding through direct contacts with the DNA. The observation that the pairwise mutation of these lysines to glutamic acid only partially eliminates the preference for supercoiled DNA suggests that other residues within this domain also contribute to the for- mation of a second DNA-binding region in the pro- tein. Taken together, the results obtained in the present study strongly support the node binding hypothesis to explain the preference of human topo- isomerase I for supercoiled DNA [36]. The related type IB topoisomerase from vaccinia virus also preferentially binds to node structures in duplex DNA [36,55]. In a recent study, it was found that the vaccinia topoisomerase binds cooperatively to DNA to form long filaments in a reaction that is nucleated by the formation of an intramolecular node on DNA [56]. Although it is not known whether the initial node binding event involves a monomer or dimer of the enzyme, if a monomer is sufficient for Supercoil binding by topoisomerase I Z. Yang et al. 5914 FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS node binding, then a second DNA-binding region must exist within the viral enzyme, as we have suggested above for the human enzyme. If this were to be the case, it is noteworthy that the structural similarity between the human and vaccinia enzymes is confined to the region referred to as subdomain III in the human enzyme [57,58] and that two of the residues in the human enzyme that we have implicated in node binding (Lys466 and Lys549) are conserved in the viral enzyme (Fig. 9). Thus, it is conceivable that the struc- tural basis for node binding by the two enzymes is similar. Experimental procedures Generation of baculovirus constructs expressing mutant proteins pFASTBAC1-topo70 K299E ⁄ S306E, pFASTBAC1-topo70 K299E ⁄ S306E ⁄ Y723F, pFASTBAC1-topo70 H266E ⁄ K299E ⁄ S306E and pFASTBAC1-topo70 H266E ⁄ K299E ⁄ - S306E ⁄ Y723F were generated as follows. The plasmid pGEX-topo70 [14] was the template for making site-directed mutations using the QuickChangeÔ mutagenesis kit from Stratagene (La Jolla, CA, USA). A pair of oligonucleotides containing the nucleotide changes for replacing Lys299 and Ser306 with glutamic acid was used to generate pGEX-topo70 K299E ⁄ S306E. The resulting plasmid and another set of oligonucleotides that changed His266 to glutamic acid were similarly used to generate pGEX-topo70 H266E ⁄ K299E ⁄ S306E. Both pGEX-topo70 K299E ⁄ S306E and pGEX- topo70 H266E ⁄ K299E ⁄ S306E were digested with NdeI and NheI and the fragments that contain the point mutations were purified and used to replace the corresponding fragments in NdeI and NheI digested pFASTBAC1-topo70 [59]. The result- ing constructs, pFASTBAC1-topo70 K299E ⁄ S306E and pFASTBAC1-topo70 H266E ⁄ K299E⁄ S306E, were used to generate baculoviruses with the Bac-to-Bac system (Invitro- gen, Carlsbad, CA, USA) in accordance with the manufac- turer’s instructions. Recombinant baculovirus infection of Sf9 cells was used to produce proteins referred to as topo70 cap- KS-E and topo70 capHKS-E, respectively. These same two pFASTBAC1 constructs were also digested with NdeI and PpuMI and the fragments containing the mutations were puri- fied by gel electrophoresis. The isolated fragments were used to replace the corresponding fragment of pFASTBAC1- topo70 Y723F [59] that had been digested with the same two restriction enzymes to generate pFASTBAC1-topo70 K299E ⁄ S306E ⁄ Y723F and pFASTBAC1-topo70 H266E ⁄ K299E ⁄ S306E ⁄ Y723F. The catalytically inactive proteins expressed in baculoviruses from these two constructs are referred to as topo70 capKS-E ⁄ Y723F and topo70 capHKS-E ⁄ Y723F, respectively. Starting from pFASTBAC1-topo70, two sets of oligonu- cleotide pairs were used to introduce clustered mutations in the linker-coding region to produce pFASTBAC1-topo70 K650A ⁄ K654A ⁄ Q657A and pFASTBAC1-topo70 K679S ⁄ K682S ⁄ K687S ⁄ K689S using the QuickChange method Fig. 9. Sequence alignment within core subdomain III of representative eukaryotic members of the type IB subfamily of topoisomerases. Human, Drosophila, Saccharomyces cerevisiae and vaccinia virus topoisomerase I sequences were aligned using CLUSTALW2 software avail- able online from the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/clustalw2/). The homology of the bacterial type IB enzymes to these eukaryotic members of the family was too weak for them to be included in the alignment. The key conserved active site residues Arg488 and Lys532 (human numbering) are marked with closed circles. The open circles identify the residues in the human enzyme (Lys466, Lys468, Lys545 and Lys549) that are implicated in the preferential binding to supercoils. Z. Yang et al. 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Acknowledgements This work was supported by Grants GM60330 and GM49156 from the National Institutes of Health We thank Matthew Redinbo and Wim Hol for their assistance with the structural comparison of core subdomain II of human topoisomerase I with homeodomains Supercoil binding by topoisomerase I We gratefully acknowledge Sharon Schultz and Heidrun Interthal for critically reading the manuscript References... gene Nucleic Acids Res 17, 8495–8509 24 Stewart AF, Herrera RE & Nordheim A (1990) Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities Cell 60, 141–149 25 Kretzschmar M, Meisterernst M & Roeder RG (1993) Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II Proc . preferential binding of human topoisomerase I to supercoiled DNA is unknown but, if node recognition is important, then it is likely that the binding involves an interaction with two regions of DNA. subdomain III. The results obtained implicate the linker and solvent-exposed basic residues in core subdomain III in the preferential binding of human topoisomerase I to supercoiled DNA. Abbreviations Dcap,. second DNA -binding site, or that the linker or basic residues in core subdo- main III are involved in the preferential binding to supercoiled DNAs. When putative DNA contact points within core subdomain II