Interaction of Human Nuclear Topoisomerase I with Guanosine Quartet-forming and Guanosine-rich Single-stranded DNA and RNA Oligonucleotides*

Human nuclear DNA topoisomerase I (top1) plays a crucial role in DNA replication, transcription, and chromosome condensation. In this study, we show that intra-and intermolecular guanosine quartets (G-quartets) can inhibit top1-mediated DNA cleavage at a high affinity site. Top1-mediated DNA cleavage was also inhibited by a 16-mer single-stranded oligodeoxynucleotide (ODN) containing a G-rich sequence (G 2 T 2 G 5 TG 2 TG 3 ) and by its RNA equivalent, neither of which form G-quartet structures. A comparison of various single-stranded ODN for their ability to inhibit top1-mediated DNA cleavage indicated that G-rich sequences containing repeats of 2 or 3 consecutive guanines interspaced with thymines spe-cifically inhibited top1. We also found that both single-stranded and G-quartet-forming ODNs bind to top1 without being cleaved by the enzyme. These results demonstrate that either DNA or RNA G-rich single-stranded and G-quartet-forming oligonucleotides can bind to top1 and prevent cleavage of duplex DNA. DNA sequences containing a high percentage of guanine (G) can form four-stranded structures referred to as G-quartets or G-quadruplexes (for review see Ref. 1). in TN5 insect cells (HighFive, Invitrogen) the recombinant baculovirus purified via the N-terminal His tag as described Oligonucleotide Labeling and Annealing— 3 (cid:2) -End-labeling was performed using terminal deoxynucleotidyl transferase (Invitrogen) with [ (cid:2) - 32 P]cordycepin 5 (cid:2) -triphosphate as described previously (39). The oligonucleotides were annealed in a buffer containing 10 m M Tris-HCl, pH 7.5, and 5 m M MgCl 2 by heating for 5 min at 95 °C and cooling 30 min at 37 °C. Intramolecular G-quartet Formation— Intramolecular G-quartet structures were prepared as described previously (15). Briefly, oligonu- cleotides at 5 (cid:3) M strand equivalent were heated at 90 °C for 5 min in 20 m M Li 3 PO 4 , pH 7.0 and then cooled at 4 °C for 30 min in the presence of 10 m M KCl. Intramolecular G-quartet formation was checked by thermal denaturation and circular dichroism experiments (15). Further dilutions of the intramolecular G-quartet oligonucleotides were performed in 10 m M KCl. Intermolecular G-quartet Formation— Intermolecular G-quartet structures were prepared as described previously (19) with the follow- ing modifications. 20 n M of 5 (cid:2) -end labeled single-stranded oligonucleotide was incubated for 72 h at room temperature with the same unla- beled strand (10 m M ) in a buffer containing 10 m M Tris-HCl, pH 7.5, and 1 M NaCl. Intermolecular G-quartet formation was checked by gel shift assay (19). Samples were diluted 1:10 with 10 m M Tris-HCl, pH 7.5, 10 m M KCl, and 1 m M EDTA prior to the addition of 0.5 volume of nondenaturing dye (30 m M Tris-HCl,

Eukaryotic nuclear DNA topoisomerase I (top1) 1 is an ubiquitous and multifunctional enzyme that regulates DNA topology during transcription, replication, chromosome condensation, and possibly recombination (for review see Refs. 21 and 22) by reversibly cleaving one strand of duplex DNA and relaxing DNA supercoiling. For the cleavage reaction, top1 forms a covalent 3Ј-phosphotyrosyl bond between a tyrosine residue (Tyr-723 for human top1) and the 3Ј-end of the broken DNA strand while leaving a 5Ј-hydroxyl terminus at the other end of the cleaved DNA (23). Under normal conditions, the cleavage complexes are rapidly reversible after relaxation of DNA supercoiling (intramolecular reactions) and eventually DNA recombination (intermolecular reactions). Top1 inhibitors, such as camptothecin and its derivatives, selectively inhibit eukaryotic top1 by reversibly preventing the religation step of the enzyme-mediated DNA nicking-closing reaction (24,25). Dissociation of the DNA strand 3Ј from the cleavage site, due to the presence of a nick, gap, or single-stranded branch in the vicinity, prevents the religation step of top1 catalytic reaction (26) and leads to irreversible cleavage complexes (27). Such substrates, commonly referred to as "suicide substrates," have been very useful in the study of the top1 catalytic cycle because they can be used to monitor, under single turnover conditions, the top1-mediated DNA cleavage step (28,29).
Because top1 has been reported to bind to noncanonical DNA structures including three-stranded flaps, triple helical DNA, and Holliday junctions (30 -34), we investigated the effect of G-quartets and various G-rich nucleic acids on top1 activity using purified recombinant human top1. A partially singlestranded 18-mer ODN, TH-18 (Fig. 1B), containing a high affinity site (26,35) (Fig. 1A) was used as a "suicide substrate" (36). In this case, top1 becomes irreversibly bonded to the 3Ј-DNA terminus, because a pentamer oligonucleotide (28,29) dissociates and therefore cannot be religated. This incomplete reaction leads to a "suicide complex" (Fig. 1B).

EXPERIMENTAL PROCEDURES
Chemicals and Enzymes-High pressure liquid chromatography-purified oligonucleotides were purchased from Bioserve Biotechnologies (Laurel, MD) and high pressure stopped-flow-purified oligonucleotides from MGW Biotech Inc. (High Point, NC). [␣-32 P]Cordycepin 5Ј-triphosphate was purchased from PerkinElmer Life Sciences. Polyacrylamide gel solutions were from National Diagnostics (Atlanta, GA). Human N-terminal truncated recombinant top1 (68 kDa) was expressed and purified essentially as reported previously (37). Briefly, the top1 gene was cloned into a baculovirus transfer vector (pBacGus-1, Novagen, Madison, WI) and used to make a recombinant baculovirus following the manufacturer's recommendations (BD-PharMingen, San Diego, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  CA). Top1 protein was then expressed in TN5 insect cells (HighFive, Invitrogen) by the recombinant baculovirus and purified via the Nterminal His tag essentially as described (26,38).
Intramolecular G-quartet Formation-Intramolecular G-quartet structures were prepared as described previously (15). Briefly, oligonucleotides at 5 M strand equivalent were heated at 90°C for 5 min in 20 mM Li 3 PO 4 , pH 7.0 and then cooled at 4°C for 30 min in the presence of 10 mM KCl. Intramolecular G-quartet formation was checked by thermal denaturation and circular dichroism experiments (15). Further dilutions of the intramolecular G-quartet oligonucleotides were performed in 10 mM KCl.
Intermolecular G-quartet Formation-Intermolecular G-quartet structures were prepared as described previously (19) with the following modifications. 20 nM of 5Ј-end labeled single-stranded oligonucleotide was incubated for 72 h at room temperature with the same unlabeled strand (10 mM ) in a buffer containing 10 mM Tris-HCl, pH 7.5, and 1 M NaCl. Intermolecular G-quartet formation was checked by gel shift assay (19). Samples were diluted 1:10 with 10 mM Tris-HCl, pH 7.5, 10 mM KCl, and 1 mM EDTA prior to the addition of 0.5 volume of nondenaturing dye (30 mM Tris-HCl, pH 7.5, 10 mM KCl, 1 mM EDTA, 1 mg/ml bromphenol blue, and 30% glycerol). The samples were then loaded on an 8% polyacrylamide native gel (29:1) run in 0.5ϫ TBE (100 mM Tris-HCl, pH 8.4, 90 mM boric acid, and 1 mM EDTA) and 10 mM KCl. Electrophoresis was performed at 4°C and 5 V/cm for 3 h.
Top1 Suicide Reaction-The suicide cleavage reactions were performed as described previously (36). Briefly, 5 pmol of recombinant top 1 were preincubated with the G-rich oligonucleotide for 5 min at room temperature in a buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 g/ml of bovine serum albumin, and 0.2 mM dithiothreitol. Irreversible top1-mediated DNA cleavage reaction was initiated by addition of 25 fmol of 3Ј-end labeled TH-18 DNA substrate in a total volume of 10 l, and the samples were incubated for 15 min at room temperature. Reactions were stopped by adding 0.5 volume of denaturing loading dye (100% formamide, 1 mg/ml bromphenol blue, and 1 mg/ml xylene cyanol blue), and the samples were loaded on a 20% (19:1) polyacrylamide gel containing 7 M urea and 1ϫ TBE.
CPT-induced DNA Cleavage Sites in the pSK Fragment-A 202-bp DNA fragment was generated by PCR from the pBluescript SK(Ϫ) phagemid (Stratagene, La Jolla, CA) using the following primers: forward (514 -534), 5Ј-GCCTCTTCGCTATTACGCCAG-3Ј; and reverse (693-716), 5Ј-GGCTGCAGGAATTCGATATCAAGC-3Ј. The PCR product was gel-purified using the QIAEX II Gel Purification System (Qiagen, Valencia, CA) and digested by HindIII. The resulting 177-bp DNA fragment was 3Ј-end labeled as described previously (40), resuspended in water, and used at ϳ50 nmol/reaction (50,000 dpm). Reaction conditions were identical to the ones used in the suicide reaction except for the addition of camptothecin (see above).
Top1 Binding Assay-10 pmol of recombinant top 1 was incubated for 30 min at room temperature with 25 fmol of 3Ј-end-labeled DNA substrate in a buffer containing 50 mM Tris-HCl, pH 7.5, and 50 mM NaCl in a total volume of 10 l. The samples were treated with 0.5 volume of nondenaturing loading dye (30 mM Tris-HCl, pH 7.5, 1 mg/ml bromphenol blue, and 30% glycerol), loaded on a 6% (19:1) polyacrylamide gel containing 0.5ϫ TBE, and electrophoresed at 4°C, 100 V for 2 h.
Imaging and quantitation of the gels were performed using a Phos-phorImager (Molecular Dynamics, Sunnyvale, CA).

Intramolecular G-quartets Inhibit top1-mediated DNA
Cleavage-We first studied the influence of two well characterized G-quartet-forming ODNs (T30923 and T40216) (15) on top1-mediated DNA cleavage ( Fig. 2A). Both oligonucleotides inhibited top1 cleavage activity at low nanomolar concentrations ( Fig. 2A). The oligonucleotide T40216 was more potent than T30923, suggesting that the length of the quartet could play a role in the inhibitory effect (Fig. 2B). T40216 was only 3 to 4 times less potent (Fig. 2B) than the high affinity full duplex substrate TH-36 (Fig. 1A), which competed with the labeled suicide substrate (final concentration 2.5 nM) and inhibited 50% of top1 cleavage (defined as IC 50 ) at ϳ6 nM (Fig. 2B). These results demonstrate that top1-mediated DNA cleavage can be inhibited at low nanomolar concentrations by intramolecular G-quartet ODNs.
Intermolecular G-quartets Inhibit top1-mediated DNA Cleavage-Because we observed that top1 can be inhibited by intramolecular G-quartets, we next investigated the effect of intermolecular G-quartet structures on top1-mediated DNA cleavage. We chose a 30-mer G-rich single-stranded oligonucleotide [ODN-1] (Fig. 3A) derived from the yeast telomeric sequence of Saccharomyces cerevisiae. This oligonucleotide can form an intermolecular parallel G-quartet (19) for which formation can be monitored by gel electrophoresis (Fig. 3B). Both single-stranded [ODN-1:SS] and quadruplex [ODN-1:G4] conformations were tested (Fig. 3C). The intermolecular G-quartet inhibited top1-mediated DNA cleavage with an IC 50 of ϳ100 nM, which is higher than for the intramolecular G-quartets T30963 and T40216 (2-and 5-fold, respectively, compare Figs. 3B and 2B). Interestingly, ODN-1:SS was also active with an IC 50 ϳ3 times higher than ODN-1:G4 (Fig. 3, B and C). These results demonstrate that intermolecular G-quartets can also inhibit top1-mediated DNA cleavage at a high affinity site in duplex DNA. . This ODN is annealed to a 36-mer complementary strand. Top1 recognizes its high affinity site, cleaves the upper strand, and liberates a 5-mer 3Ј-end-labeled ODN that separates from the uncleaved strand on a denaturing gel. Top1 remains bound at the 3Ј extremity of the cleaved strand (suicide complex).
A Single-stranded 16-mer G-rich ODN Inhibits top1-mediated DNA Cleavage-We next investigated whether a G-quartet-forming structure or a sequence containing consecutive guanosines (G-stretch) was sufficient to prevent top1 cleavage at its high affinity site. For this purpose, we designed the oligonucleotide [ODN-2], which was derived from T30923 (Fig.  4A). ODN-2 has the same base content as T30923 (12 guanines and 4 thymines) but cannot form either intra-or intermolecular G-quartets as verified by gel mobility, thermal denaturation, and circular dichroism experiments (data not shown). Nevertheless, ODN-2 inhibited top1-mediated DNA cleavage at low nanomolar concentration (Fig. 4B) and was as potent as the intramolecular G-quartets T30963 and T40216 (see Fig. 2). ODN-2 was ϳ6-fold more efficient than its duplex homologue, , and was markedly more potent than its C-rich complementary strand, [ODN-2c] (by at least 2 orders of magnitude) (Fig. 4B). ODN-2 was also ϳ50-fold more potent than a non-G-rich single-stranded DNA of the same length, corresponding to the last 16 bases of HIV-1 U5 long terminal repeat sequence . These results demonstrate that the singlestranded G-rich oligonucleotide ODN-2 effectively inhibits DNA cleavage.
DNA Sequence Specificity of G-rich Single-stranded ODNs-To further investigate the influence of the guanine distribution in single-stranded oligonucleotides, different Grich sequences were tested (Fig. 5A). In the oligonucleotide ODN-5, one of the thymines was moved in order to disrupt the G-stretch present in the middle of ODN-2 (Fig. 5A). ODN-5 showed approximately the same potency as ODN-2 (Fig. 5B), indicating that the 5-mer G-stretch present in the middle of ODN-2 is not required for top1 inhibition. Consistent with this observation, we found that an oligonucleotide containing only a central stretch of guanosines flanked by one thymine, [ODN-10], only inhibited top1-mediated DNA cleavage above micromolar concentrations (Fig. 5B).
The presence and position of the thymines in ODN-2 were then investigated by placing two of the four thymines at both extremities of the sequence. ODN-6 was ϳ5-fold less potent than ODN-2 (Fig. 5B). This difference increased to ϳ15-fold when the thymines were replaced by four guanines in the poly-G ODN-7, demonstrating the importance of the thymines for activity.
To investigate whether top1 cleavage could be inhibited by 5Ј-TpG-3Ј steps, which represent high affinity sites for human top1 in duplex DNA (28,41,42), a 16-mer ODN with an alternating TG sequence, , was tested. This oligonucleotide was ϳ150-fold less potent than ODN-2. Weak activity was also observed for a 16-mer oligonucleotide formed by four TTGG repeats, [ODN-9]. These results demonstrate the importance of the DNA sequence of G-rich single-stranded ODNs for top1-mediated DNA cleavage inhibition.

G-quartet-forming and G-rich Single-stranded ODNs Inhibit
Camptothecin-induced top1 Trapping on DNA-We next tested the effect of G-rich ODNs on camptothecin-induced top1 trapping on DNA. We performed these experiments with the PvuII-HindIII digested pBluescript SK(Ϫ) 177-bp DNA fragment, which contains previously characterized top1 cleavage sites (40). Fig. 6 shows that both oligonucleotides T30923 and ODN-2 inhibited the camptothecin-induced DNA cleavage at all sites examined. As in the case of the TH-18 suicide substrate, ODN-2 was more potent than T30923 (Fig. 6).
G-rich Single-stranded and G-quartet-forming ODNs Both Bind to top1-Gel shift experiments were performed to determine top1 binding to the G-rich single-stranded ODN-2 and to the G-quartet forming oligonucleotides (T30923 & T40216). These three substrates produced a band with retarded electrophoretic mobility when incubated with purified top1 (Fig. 7A). Binding efficiencies for these oligonucleotides were lower than for TH-36.
The G-rich Single-stranded ODN-2 Displaces top1 from Its High Affinity Substrate-To investigate further the binding of G-rich oligonucleotides to top1, competition experiments were performed in the presence of both the high affinity duplex TH-36 and ODN-2. In these experiments, a DNA-protein complex was formed between top1 and 32 P-labeled TH-36 in the presence of increasing concentrations of unlabeled ODN-2 or unlabeled TH-36 (Fig. 7B). At high concentrations of unlabeled oligonucleotide, the band corresponding to the top1-labeled TH-36 complex on the gel disappeared for both ODN-2 and TH-36. This indicates that both oligonucleotides displaced top1 from its labeled substrate (TH-36). The efficiency of strand displacement was ϳ10 times lower for ODN-2 than for TH-36 (Fig. 7B). Furthermore, in the experiments including both TH-36 and ODN-2, we did not observe any supershift of the  6. Inhibition of camptothecin-induced top1 cleavage by G-quartet and G-rich single-stranded ODNs. The 177-bp pBluescript SK(Ϫ) fragment was generated by PCR and labeled as described previously (40). Camptothecin (CPT) was used at 10 M to block top1mediated religation and therefore reveal top1 cleavage sites. top1-DNA band suggesting that top1 binds either to its duplex high affinity substrate TH-36 or to the single-stranded G-rich ODN-2 but not to both at the same time. These results demonstrate that G-rich oligonucleotides can bind competitively to top1 and prevent the enzyme from cleaving duplex DNA.
G-quartet-forming and G-rich Single-stranded ODNs Are Not top1 Substrates-To determine whether the G-quartets and G-rich single-stranded ODNs could be substrates for top1, the G-rich ODNs were labeled and tested for top1-mediated DNA cleavage. As expected, camptothecin induced cleavage in the TH-36 ODN (see Fig. 1A for sequence). Camptothecin also enhanced cleavage in the suicide TH-18 ODN, indicating that top1-mediated DNA cleavage in this oligonucleotide is partially reversible. Using ODN-2, cleavage was not detected either in the absence or presence of camptothecin (Fig. 8). A similar result was obtained with the G-quartet ODNs T30923 and T40216 (data not shown), demonstrating that these G-rich ODNs are not cleaved by top1.
The RNA Equivalent of ODN-2 Also Inhibits top1-mediated DNA Cleavage-We next tested the effect of single-stranded RNA on the inhibition of top1-mediated DNA cleavage. First we designed an oligodeoxynucleotide identical to ODN-2 in which thymines were replaced by uracils (Fig. 9A). ODN-11 inhibited top1 DNA-mediated cleavage (Fig. 9B) and was ϳ4-fold less potent than the parental ODN-2. The RNA equivalent of ODN-11,   (Fig. 9A), was ϳ2-fold more potent than its ODN homologue (Fig. 9, B and C). Thus, top1-mediated DNA cleavage can be efficiently inhibited by single-stranded G-rich RNA. DISCUSSION The present study demonstrates that both intra-and intermolecular G-quartets and single-stranded G-rich ODNs inhibit top1-mediated DNA cleavage. This inhibition appears to re- quire repeats of at least 2ϳ3 consecutive guanines interspaced with thymines. We also demonstrate that the inhibition of top1-mediated DNA cleavage can be efficiently achieved with G-rich RNA and that the G-quartet-forming and singlestranded G-rich ODNs bind competitively to top1 without being cleaved by the enzyme.
It is becoming increasingly clear that top1 can bind to noncanonical DNA structures. DNA containing partially singlestranded regions and extra-helical segments (bulges) were shown to be cleaved by the enzyme (26,43,44). Top1 also cleaves DNA sites with local structure distortion including oxidative base modifications (36), base mismatches and abasic sites (45), UV photoproducts (46), base methylation (47), intercalation (48), and minor groove alkylation (49,50). Top1 has also been found to cleave three-stranded flap regions and promote their DNA recombination (30,31). Top1 binding to triple helical DNA has also been reported (32) as well as binding to Holliday junctions, which correspond to four-stranded structures as in the case of G-quartets (33,34). However in contrast to the Holliday junctions, the G-quartets we examined are not cleaved by top1. More recently, Hélène and co-workers (51) also reported that top1 binds to preformed inter-and intramolecular G-quartets and promotes the opening of a 69-bp duplex to form a stable intermolecular G-quartet-containing structure. It is therefore intriguing how top1 can bind to such diverse structures. Recently the structure of top1 bound covalently and noncovalently to duplex DNA has been determined, and the enzyme has been shown to encircle the DNA duplex (52,53). The finding that top1 can also bind to G-rich single-stranded and four-stranded DNA suggests that the enzyme can accommodate various size nucleic acids.
High affinity binding of top1 to single-stranded DNA or RNA has not been reported previously. In fact, binding to nonspecific single-stranded DNA with random sequence was weak (54) and was detectable only in segments that had the potential to form duplex structures (55). Top1 is known to play an important role in transcription (for review see Ref. 56) by direct binding to transcription complexes (57-60) and phosphorylation of serinearginine rich (SR) splicing factors that control pre-mRNA splicing (61). Top1 enhances TFIID-TFIIA complex assembly during activation of transcription (62) and interacts with TFIIIC to promote accurate termination and reinitiation (63). In this work, we report top1 inhibition by G-rich-containing RNA oligonucleotides. The in vivo significance of top1 binding to T-and G-rich single-stranded RNA still remains to be determined, but it is possible that top1 could play a role in the stabilization of such sequences during transcription, preventing their folding into higher order structures. Top1 binding to T-and G-rich single-stranded RNA might contribute to the activity of the enzyme in RNA splicing (64,65). These results point to top1 being a multifunctional enzyme capable of accommodating varied DNA structures.