Mutation of a Conserved Serine Residue in a Quinolone-resistant Type II Topoisomerase Alters the Enzyme-DNA and Drug Interactions*

A Ser740 → Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2S740W in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2S740W induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position −2 and G at position +6 were observed for the top2S740W in addition to the previously reported C−1 and G+5 for the wild-type top2. The VP-16-induced top2S740Wcleavage complexes were also more stable. The most stable sites had strong preference for C−1, whereas the most reversible sites showed no base preference at positions −1 or −2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser740→ Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.

DNA topoisomerases are enzymes that catalyze changes in the topology of DNA via a mechanism involving the transient breakage and rejoining of phosphodiester bonds in the DNA backbone (1). Studies in both prokaryotic and eukaryotic cells have demonstrated the importance of topoisomerases in transcription, DNA replication, and chromosome segregation. The type II topoisomerases, which make transient double-strand breaks and change the linking number of DNA in steps of two, play key roles in chromosome structure. In eukaryotic cells, these enzymes are essential for chromosome condensation/decondensation and decatenation of chromosome loops during mitosis (2,3).
In Escherichia coli, mutations that lead to quinolone resistance are most often found in gyrA, the structural gene for the DNA gyrase A subunit. Ser 83 of gyrA is the amino acid most frequently changed in strains with high levels of quinolone resistance, although other mutations in either gyrA or gyrB can lead to quinolone resistance (15). Mutations that change Ser 83 to either leucine or tryptophan confer high levels of quinolone resistance, whereas changing Ser 83 to alanine results in a low level of quinolone resistance (16).
A previous study examined the effects of yeast top2 mutations that change Ser 740 (numbering of amino acid residues was corrected according to Ref. 17; Ser 740 was previously referred to as Ser 741 (18)), the amino acid homologous to Ser 83 in gyrA of E. coli (19). A mutation changing Ser 740 3 Trp resulted in resistance to CP-115,953 and hypersensitivity to etoposide (19). To investigate the basis for the differential responses of the top2 S740W , we analyzed the base sequence preference (20 -23) and stability of the top2 cleavage complexes in the absence and presence of top2-targeting drugs.  740 3 Trp mutation in yeast top2 alters the enzyme-mediated DNA cleavage sites in the absence of inhibitors. A 254-base pair DNA fragment from the first c-myc intron was prepared between positions 3035 and 3288 by PCR using one primer labeled with 32 P at the 5Ј-terminus. Panel A, labeling of the upper DNA strand at position 3035. Panel B, labeling of the lower DNA strand at position 3288. Top2 reactions were performed at 37°C for 30 min in the presence of 5 mM MgCl 2 or 5 mM CaCl 2 as indicated and stopped by adding EDTA and SDS (25 mM and 1% final concentrations, respectively). The purine ladder was obtained after formic acid reaction. yWT, yeast wild-type top2; yS740W, top2 S740W . Double-headed arrows correspond to DNA cleavage sites with a 4-base pair stagger which represent potential DNA double-strand breaks.
Preparation of End-labeled DNA Fragments by PCR-Three sets of labeled DNA fragments were prepared from the human c-myc gene by PCR. A 254-base pair DNA fragment from the first intron was prepared between positions 3035 and 3288, with numbers referring to GenBank genomic positions using oligonucleotides 5Ј-GTAATCCAGAACTG-GATCGG-3Ј for the upper strand and 5Ј-ATGCGGTCCCTACTC-CAAGG-3Ј for the lower strand (annealing temperature, 56°C). A 401base pair DNA fragment from the junction between the first intron and first exon was prepared between positions 2671 and 3072 using oligonucleotides 5Ј-TGCCGCATCCACGAAACTTT-3Ј for the upper strand and 5Ј-TTGACAAGTCACTTTACCCC-3Ј for the lower strand (annealing temperature, 60°C). A 480-base pair fragment from the first exon containing promoters P 1 and P 2 was prepared between positions 2265 and 2745 using the oligonucleotides: 5Ј-GATCCTCTCTCGCTAATCTC-CGCCC-3Ј for the upper strand and 5Ј-TCCTTGCTCGGGTGTTGTA-AGTTCC-3Ј for the lower strand (annealing temperature, 70°C). Single-end labeling of these DNA fragments was obtained by 5Ј-end labeling of the adequate primer oligonucleotide. 10 pmol of DNA was incubated for 60 min at 37°C with 10 units of T4 polynucleotide kinase and 10 pM [␥-32 P]ATP (100 Ci) in kinase buffer (70 mM Tris-HCl, pH 7.6, 0.1 M KCl, 10 mM MgCl 2 , 5 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin). Reactions were stopped by heat denaturation at 70°C for 15 min. After purification using Sephadex G-25 columns (Boehringer Mannheim), the labeled oligonucleotides were used for PCR. Approximately 0.1 g of the c-myc DNA that had been restricted by SmaI and PvuII (fragment 2265-2745), XhoI and XbaI (fragment 2671-3072 and fragment 3035-3288) was used as template for the PCR. 10 pmol of each oligonucleotide primer, one of them being 5Ј-labeled, was used in 22 temperature cycle reactions (each cycle with 94°C for 1 min, annealing for 1 min, and 72°C for 2 min). The last extension was for 10 min. DNA was purified using PCR Select-II columns (5Prime-3Prime, Inc. Boulder, CO).
Overexpression and Purification of Yeast Top2-Wild-type yeast top2 and Ser 740 3 Trp proteins were overexpressed using YEpTOP2-PGAL1 or YEptop2-S*W-PGAL1 using yeast strain JEL1t1 Ϫ (24) and purified to homogeneity as described previously (25). The detailed procedure has been described elsewhere (26). Top2 reactions were carried out as reported (26,27) using either supercoiled pBR322 to monitor ATP-dependent relaxation or kinetoplast DNA isolated from Crithidia fasciculata to monitor decatenation.
Calcium-promoted DNA cleavage was performed in the same buffer with 5 mM CaCl 2 instead of MgCl 2 . Reactions were performed at 37°C for 30 min and thereafter stopped by adding 1% SDS and 0.4 mg/ml proteinase K (final concentrations) followed by an additional incubation at 55°C for 30 min.
Electrophoresis and Base Preference Analysis-For DNA sequence analysis, samples were precipitated with ethanol and resuspended in 5 l of loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). Samples were heated to 95°C for 5 min and thereafter loaded onto DNA sequencing gels (7% polyacrylamide; 19:1 acrylamide/bisacrylamide) containing 7 M urea in 1 ϫTris borate/EDTA buffer. Electrophoresis was performed at 2,500 volts (60 watts) for 2-3 h. The gels were dried on Whatman No. 3MM Position 0 corresponds to the cleavage site. Panels A and B, probability of the observed base frequency deviations from expectation. In the y axis, p is the probability of observing that deviation or more, either as excess (above base line) or deficiency (below base line) relative to the expected frequency of each individual base (20,21). Panel A, all cleavage sites were analyzed. Panel B, only specific cleavage sites were analyzed. Panel C, base distribution at each position. Underlined numbers represent base frequencies significantly (p Ͻ 0.001) greater or lower than expected.
FIG. 3-continued paper sheets and visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. The determination of preferred bases around top2 cleavage sites was done as described previously (20,21).

Mapping and Analysis of the Cleavage Sites Induced by Mutant and Wild-type Top2 in the Presence of Different Top2
Poisons-The cleavage sites induced by the top2 S740W protein in the presence of various top2 poisons were mapped in the upper and lower strands of a fragment of the human c-myc gene. This DNA fragment includes the junction between the first exon and first intron (28). Fig. 1 presents the cleavage pattern obtained in the presence of etoposide, azatoxin, the fluoroquinolone CP-115,953, and the intercalating agents amsacrine and mitoxantrone. The top2 S740W protein was characterized previously as partially resistant to fluoroquinolones; and when compared with the wild-type enzyme, several cleavage sites induced in the presence of CP-115,953 were reduced markedly. The result with amsacrine is particularly interesting because the wild-type and top2 S740W protein have similar sensitivities to amsacrine in vivo (25). Taken together, these results show that the Ser 740 3 Trp mutation affects the DNA cleavage patterns induced by both intercalating and nonintercalating drugs.
Calcium-promoted DNA Cleavage Sites Differ between the Top2 S740W and the Wild-type Enzyme-To investigate whether the altered DNA cleavage activity of the top2 S740W was drugdependent, we compared the calcium-promoted DNA cleavage (29) for the Ser 740 3 Trp protein and the wild-type enzyme in the two strands of the c-myc first intron fragment (Fig. 2). Even in the presence of magnesium, differences in the cleavage patterns could be observed. When magnesium was replaced by calcium, higher levels of DNA cleavage were seen with both proteins. DNA cleavage sites common to both proteins were seen in the presence of Ca 2ϩ ; however, there were also major differences in the intensity of cleavage at other sites. Most of the sites of DNA cleavage in the upper and lower strands were staggered by 4 base pairs with a 5Ј-overhang, as expected for concerted top2-induced double-strand cleavage (2,5,7). These results suggest that the top2 S740W mutation also alters DNA cleavage in the absence of a topoisomerase II poison.
Altered Base Preference of the Etoposide-stabilized Cleavage Complexes for the Mutant Top2 S740W -As described above, the top2 S740W protein is hypersensitive to etoposide. It was therefore of considerable interest to examine the effect of this mutation on the DNA base preference of top2 in the presence of this drug (Fig. 3). Cleavage sites for three c-myc DNA fragments (see "Experimental Procedures") were analyzed for both DNA strands. For the wild-type top2 protein, etoposide preferentially stabilized sites with CϪ1 (103 out of 167 sites) (Fig.  3C). This result agrees well with previous analyses of cleavage of the same DNA fragments by human top2 in the presence of etoposide (5,30). Preference on the opposite strand showed a complementary (although slightly weaker) preference for Gϩ5. Top2 S740W also demonstrated a strong preference for CϪ1. In addition, a novel preference for C at position Ϫ2 (94 out of 176 sites) in combination with the complementary G at position ϩ6 (84 out of 176 sites) was also seen. To focus on the impact of the Ser 740 3 Trp mutation on base preference, we analyzed separately those DNA cleavage sites exclusively detectable for top2 S740W or for the wild-type enzyme (Fig. 3B). These unique DNA cleavage sites did not show any clear CϪ1 or Gϩ5 pref- erence for either protein, which is expected because both proteins have a CϪ1/Gϩ5 preference. However, top2 S740W still showed a significant preference of C at position Ϫ2 (45 out of 63 sites) in combination with a preference for the complementary G at position ϩ6 (39 out of 63 sites). In addition, a chi-square test indicated that the combination of the CϪ1 and CϪ2 preference in the top2 S740W was not significantly more frequent than having CϪ1 or CϪ2 alone. Thus, the novel CϪ2 base preference in the top2 S740W is independent of the CϪ1 preference. In contrast, the cleavage sites unique for the wild-type protein tended to exclude sites with Cϩ2 (7 out of 54 sites) and with Gϩ6 (9 out of 54 sites). These data show a change in the protein-DNA interaction resulting from the Ser 740 3 Trp mutation, leading to an extension of the base preference for the CϪ2 position in the presence of etoposide.
Enhanced Salt and Heat Stability of the Cleavage Complexes Mediated by Top2 S740W in the Presence of Etoposide-The effect of the Ser 740 3 Trp mutation on the stability of specific top2⅐DNA cleavage complexes was determined by examining the salt and heat reversibility of the ternary complex formed with drug, protein, and DNA (Fig. 4). Cleavage reactions were carried out with the wild-type or the Ser 740 3 Trp top2 for 30 min at 37°C. The reactions were heated to 65°C for various times before the addition of SDS. Fig. 4 shows the result for the upper (panel A) and lower strand (panel B) of the c-myc fragment corresponding to the first intron. Most of the etoposidestabilized cleavage sites were readily reversible for the wildtype protein. In contrast, a number of cleavage sites induced by top2 S740W showed slow reversal (e.g. positions 3073, 3091, 3163, 3223, and 3183) or no detectable reversal after a 30-min incubation at 65°C (e.g. positions 3167, 3171, 3241, 3174, and 3170). Enhanced heat stability of the DNA cleavage sites induced by top2 S740W was also observed in the other c-myc DNA fragments (data not shown).
To focus on the role of etoposide-protein-DNA interactions on the stability of the DNA-enzyme interaction, the salt reversibility (0.5 M NaCl final concentration) of the calcium-promoted or etoposide-stabilized cleavage complexes was examined (Fig.  5). The calcium-promoted cleavage complexes were readily salt reversible for both top2 S740W and the wild-type enzyme (Fig.   FIG. 6. Cleavage complexes induced by top2 S740W consist predominantly of DNA double-strand breaks. The DNA fragment was the same as in Fig. 2A. The upper DNA strand was labeled at position 3035. Top2 reactions were performed at 37°C for 30 min. The reactions were then incubated at 65°C for the indicated times before the addition of SDS and proteinase K. For DNA sequence analysis, samples were precipitated with ethanol and resuspended in 5 l of loading buffer (30% glycerol, 1 mM EDTA, pH 8, 10 mM Tris, pH 7.4, and 0.1% bromphenol blue). Samples were loaded onto nondenaturing DNA sequencing gels (7% polyacrylamide; 19:1 acrylamide/bisacrylamide in 1ϫ Tris borate/EDTA buffer). Electrophoresis was performed at 45 watts for 2-3 h. Top2, no drug treatment; Control, no top2, no drug treatment; yWT, yeast wild-type top2; yS740W, top2 S740W .
FIG. 5. Cleavage complexes stabilized by 100 M etoposide with top2 S740W exhibit enhanced salt stability. The DNA fragment was the same as in Fig. 2A. Panel A, salt stability of calcium-promoted (5 mM) cleavage sites. Panel B, salt stability of 100 M etoposide-stabilized cleavage complexes.Top2 reactions were performed at 37°C for 30 min. After the addition of NaCl (0.5 M NaCl final concentration), the reactions were incubated at 37°C for the indicated times before the addition of SDS and proteinase K. Top2, no drug treatment. Numbers correspond to genomic positions of the nucleotide covalently linked to top2. yWT, yeast wild-type top2; yS740W, top2 S740W . 5A). By contrast, all etoposide-stabilized cleavages induced by top2 S740W were completely salt-stable even after 30 min. Most of the cleavage complexes reversed at least partially for the wild-type top2 (Fig. 5B). These results support previous results suggesting that etoposide more strongly stabilizes the top2⅐DNA complexes formed with the top2 S740W enzyme.
Lee and Hsieh (31) showed previously that heat or salt incompletely reversed teniposide-stabilized covalent complexes induced with Drosophila top2. They did not observe stable DNA double-strand cleavage following heat or salt reversal. To determine whether the etoposide-stabilized, heat-stable top2 S740W cleavages were predominantly DNA single-or double-strand breaks, we performed nondenaturing gel electrophoresis (Fig. 6). Several strong cleavage sites were observed on nondenaturing gels, indicating that the top2 S740W protein generates stable double-as well as single-strand breaks. Slight heat stability was also seen with the wild-type top2 mediated at sites 3171, 3175, and 3238, but the stability was consider- ably less than was seen with the top2 S740W protein.
Reversibility of the Etoposide-induced Cleavage Complexes Is Base Sequence-dependent-Because the top2 S740W exhibited a large number of cleavage sites with different reversal kinetics after heat treatment, we sequenced these sites to study the influence of local base preference on top2 religation kinetics for the top2 S740W (Fig. 7). Etoposide-stabilized cleavage sites were divided into rapidly reversible (complete reversal within 2 min at 65°C) or slowly reversible (incomplete reversal after 2 min). Cleavage sites with slow reversibility exhibited highly significant preference for CϪ1 (78 out of 89 sites) in combination with a less strong CϪ2 preference (54 out of 89 sites) (Fig. 7B). Complementary preference was observed on the opposite strand with preference (although weaker) for Gϩ5 (53 out of 89 sites) and Gϩ6 (36 out of 89 sites) as well. In contrast, rapidly reversible cleavage sites did not show any base preference at positions Ϫ1 and Ϫ2. However, they displayed a significant Gϩ5 and Gϩ6 base preference, indicating DNA cleavages in the complementary strand with a CϪ1 and CϪ2 base preference. These data are consistent with the formation of stable cleavage complexes when the preferred base(s) occur(s) on the DNA strand that is cleaved by the enzyme. Religation is influenced less by the base sequence on the opposite strand. DISCUSSION A number of factors contribute to the sensitivity of cells toward agents targeted to the type II topoisomerases (32)(33)(34)(35). Top2 mutations that alter drug-induced DNA cleavage result in marked alterations, ranging from high resistance (26,36,37) to severalfold hypersensitivity (19,26). However, the mechanisms by which mutations within the enzyme alter drug sen- sitivity have not been defined. Decreased drug binding to the top2⅐DNA complex has been reported for quinolone-resistant DNA gyrase with a Ser 83 3 Trp mutation in the A subunit (7,38). A homologous mutation in the yeast top2 gene, which changes Ser 740 into Trp, also results in quinolone resistance (19). The same region of the protein is clearly important for determining sensitivity to eukaryotic topoisomerase II poisons because the mutation also causes hypersensitivity to etoposide (19).
The results presented in this report show that the Ser 740 3 Trp mutation in yeast top2 affects the DNA-protein interactions. In the absence of any drug, the calcium-promoted DNA cleavage sites of top2 S740W were clearly different from those induced by the wild-type enzyme, indicating a change in DNA recognition. Amino acid residue 740 is part of the ␣4 DNArecognition helix within the helix-turn-helix (HTH) motif of top2 (39). This HTH motif and its counterpart in E. coli gyrase are mutational hotspots for resistance to drugs that stabilize the cleaved state of DNA (15,39). DNA footprinting has shown that for top2 and DNA gyrase approximately 15-35 base pairs of DNA are protected by the enzyme (40,41). In addition, a 29-kDa fragment containing the active-site tyrosine and the HTH motif can be cross-linked to DNA (42), and protein footprinting has demonstrated that the presence of DNA protects the HTH motif from chemical modification (43). The data of this study are consistent with a direct interaction of the HTH motif with DNA (44). Peptides containing aromatic amino acids are known to be capable of partial intercalation with DNA (45). NMR titrations with complexes between double-strand DNA and tryptophan-containing peptides confirmed the possibility of intercalation (46). Consistent with this possibility, Fig. 8 presents the position of Ser 740 on the protein surface and its close proximity to the DNA. In addition, Ser 740 is approximately 3.0 Å from Tyr 734 , and these residues could form a hydrogen bond. Thus, the Ser 740 3 Trp mutation could alter the DNA-top2 interaction directly by intercalation as well as indirectly by changing the enzyme conformation and modifying DNA recognition.
The Ser 740 3 Trp mutation in yeast top2 also markedly affected the protein-drug interaction. We presented two lines of evidence that, compared with the wild-type protein, the increased heat and salt stability of top2 S740W -induced DNA cleavages are dependent on etoposide interaction. First, calciumpromoted cleavages, although presenting an altered DNAprotein interaction, were readily reversible. Second, cleavages on a DNA strand without the etoposide-preferred bases by cooperative effects with the other subunit are also readily reversible (30,47). The fact that a single mutation at amino acid residue 740 changed the quinolone as well as the etoposide sensitivity is consistent with previous studies suggesting that quinolones share a common interaction domain on eukaryotic top2 with other DNA cleavage-enhancing drugs (48). Based on drug-associated preferences for the bases immediately flanking the top2-linked DNA cleavage site, we also proposed a drugstacking model in which the drugs generally occupy a common site at the interface of the enzyme and the ends of the cleaved DNA (5, 20 -22).
Our data provide evidence that the Ser 740 3 Trp mutation might directly or indirectly change at least overlapping quinolone-protein and etoposide-protein interaction domains. Furthermore, we found a novel CϪ2 base preference in the top2 S740W in the presence of etoposide. This new CϪ2 preference proved to be statistically independent of the common CϪ1 preference. These findings suggest a model of a more relaxed drug binding site for the Ser 740 3 Trp enzyme allowing etoposide to interact with CϪ2 in addition to CϪ1. This model does not conflict with the hypothesis of etoposide acting at the DNAprotein interface (5,21,30) because the altered interface between the top2 S740W and its DNA substrate is likely to increase the etoposide binding affinity and hence to increase the stability of the ternary complex.
The results of this study suggest a DNA-top2 binding site on the protein surface, which is directly or indirectly affected by amino acid residue 740 and hence controls the binding of drugs including quinolones and etoposide to the top2⅐DNA complex.
Further structural studies with wild-type and Ser 740 3 Trp mutant top2 in the presence of DNA and inhibitors are awaited. A DNA fragment of the c-myc first intron between positions 3185 and 3168, containing several highly salt-and heat-stable cleavage sites, could be a suitable DNA substrate.