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

Dirk StrumbergDagger , John L. Nitiss§, Angela Rose§, Marc C. Nicklausparallel , and Yves Pommier**

From the Laboratory of Molecular Pharmacology and the parallel  Laboratory of Medicinal Chemistry, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255 and the § Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

    ABSTRACT
Top
Abstract
Introduction
References

A Ser740 right-arrow 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 top2S740W cleavage 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 right-arrow 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.

    INTRODUCTION
Top
Abstract
Introduction
References

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).

Topoisomerase II (top2)1 has also been identified as a major target of chemotherapeutic agents that are specifically active against prokaryotic (4) or cancer cells (5-8). Fluoroquinolones are antibacterial agents that target DNA gyrase (the prokaryotic type II topoisomerase) (9), whereas a variety of DNA-intercalating agents such as anthracyclines, amsacrine, and mitoxantrone and nonintercalating agents such as the epipodophyllotoxin etoposide (VP-16) are active against eukaryotic top2 and are clinically important anti-cancer agents (5-8). Azatoxin is also a nonintercalative top2 poison (10).

Recently, it has been shown that 6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid (CP-115, 953), a fluoroquinolone closely related to ciprofloxacin, is highly toxic to mammalian cells in culture (11, 12). Studies in yeast demonstrated that top2 is the primary physiological target for this quinolone (13). Unlike etoposide, which stabilizes top2- mediated DNA cleavage primarily by inhibiting the religation reaction of top2, CP-115,953 stabilizes DNA cleavage by enhancing the forward rate of cleavage (14).

In Escherichia coli, mutations that lead to quinolone resistance are most often found in gyrA, the structural gene for the DNA gyrase A subunit. Ser83 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 Ser83 to either leucine or tryptophan confer high levels of quinolone resistance, whereas changing Ser83 to alanine results in a low level of quinolone resistance (16).

A previous study examined the effects of yeast top2 mutations that change Ser740 (numbering of amino acid residues was corrected according to Ref. 17; Ser740 was previously referred to as Ser741 (18)), the amino acid homologous to Ser83 in gyrA of E. coli (19). A mutation changing Ser740right-arrow Trp resulted in resistance to CP-115,953 and hypersensitivity to etoposide (19). To investigate the basis for the differential responses of the top2S740W, we analyzed the base sequence preference (20-23) and stability of the top2 cleavage complexes in the absence and presence of top2-targeting drugs.

    EXPERIMENTAL PROCEDURES

Materials, Chemicals, and Enzymes-- Etoposide (VP-16) was obtained from Bristol-Myers Co., Wallingford, CT. Amsacrine and mitoxantrone were from the Drug Synthesis and Chemistry Branch (NCI, National Institutes of Health). Azatoxin was provided by Dr. T. Macdonald, Department of Chemistry of the University of Virginia, Charlottesville. CP-115,953 was the gift of Drs. P. R. McGuirk and T. D. Gootz of Pfizer Laboratories. Drug stock solutions were made in dimethyl sulfoxide at 10 mM. Further dilutions were made in distilled water immediately before use. Human c-myc inserted into pBR322, restriction enzymes, T4 polynucleotide kinase, polyacrylamide/bisacrylamide, and Taq DNA polymerase were purchased from Lofstrand Labs (Gaithersburg, MD), Life Technologies, Inc., New England Biolabs (Beverly, MA), or Qiagen Inc. (Valencia, CA). [gamma -32P]ATP was purchased from NEN Life Science Products. PCR oligonucleotide-primer were obtained from Life Technologies, Inc. (Gaithersburg, MD).

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 5'-GTAATCCAGAACTGGATCGG-3' for the upper strand and 5'-ATGCGGTCCCTACTCCAAGG-3' for the lower strand (annealing temperature, 56 °C). A 401-base pair DNA fragment from the junction between the first intron and first exon was prepared between positions 2671 and 3072 using oligonucleotides 5'-TGCCGCATCCACGAAACTTT-3' for the upper strand and 5'-TTGACAAGTCACTTTACCCC-3' for the lower strand (annealing temperature, 60 °C). A 480-base pair fragment from the first exon containing promoters P1 and P2 was prepared between positions 2265 and 2745 using the oligonucleotides: 5'-GATCCTCTCTCGCTAATCTCCGCCC-3' for the upper strand and 5'-TCCTTGCTCGGGTGTTGTAAGTTCC-3' for the lower strand (annealing temperature, 70 °C). Single-end labeling of these DNA fragments was obtained by 5'-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 [gamma -32P]ATP (100 µCi) in kinase buffer (70 mM Tris-HCl, pH 7.6, 0.1 M KCl, 10 mM MgCl2, 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 µg 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 5'-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 Ser740right-arrow Trp proteins were overexpressed using YEpTOP2-PGAL1 or YEptop2-S*W-PGAL1 using yeast strain JEL1t1- (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.

Top2-induced DNA Cleavage Reactions-- DNA fragments (5-10 × 104 dpm/reaction) were equilibrated with or without drug in 1% dimethyl sulfoxide, 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.1 mM Na2EDTA, 1 mM ATP, and 15 µg/ml bovine serum albumin for 5 min before the addition of 8 units (80 ng) of purified top2 in a 10-µl final reaction volume.

Calcium-promoted DNA cleavage was performed in the same buffer with 5 mM CaCl2 instead of MgCl2. 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 µl 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 ×Tris borate/EDTA buffer. Electrophoresis was performed at 2,500 volts (60 watts) for 2-3 h. The gels were dried on Whatman No. 3MM 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).

    RESULTS

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 top2S740W 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 top2S740W 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. For example, cleavage by top2S740W on the upper strand of the c-myc DNA fragment at positions 2707, 2718, 2741, 2746, and 2768 was clearly reduced. By contrast, the mutant protein caused increased cleavage at specific sites in presence of etoposide, e.g. at positions 2711, 2712, 2781, 3026, and 3019 compared with the wild-type enzyme. Azatoxin, another nonintercalating top2 poison (10), also showed enhanced DNA cleavage at several sites. It should be noted that reduced cleavage in the presence of etoposide occurs at other sites, e.g. 2839 and 2776. The intercalating agent mitoxantrone resulted in less cleavage with the top2S740W protein at sites such as 2707, 2735, 2785, 2807, 2953, and 2974, whereas at other sites, less cleavage was seen with the wild-type protein (e.g. positions 2813, 2898, 2901, and 3011). Similarly, multiple changes in the cleavage sites induced in the presence of amsacrine were seen. The result with amsacrine is particularly interesting because the wild-type and top2S740W protein have similar sensitivities to amsacrine in vivo (25). Taken together, these results show that the Ser740right-arrow Trp mutation affects the DNA cleavage patterns induced by both intercalating and nonintercalating drugs.


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Fig. 1.   Ser740 right-arrow Trp mutation in yeast top2 alters DNA cleavage patterns induced by various top2 inhibitors in the human c-myc gene. A DNA fragment from the junction between the c-myc first intron and first exon between positions 2671 and 3072 was prepared by PCR using one primer labeled with 32P at the 5'-terminus. Panel A, labeling of the upper DNA strand at position 2671. Panel B, labeling of the lower DNA strand at position 3072. Top2 reactions were performed at 37 °C for 30 min and stopped by adding SDS and proteinase K (1% and 0.4 mg/ml final concentrations, respectively). DNA electrophoresis was in 7% denaturing acrylamide gels (7 M urea) in TBE buffer. Drugs are indicated above each lane (100 µM etoposide, 100 µM CP-115,953, 200 µM azatoxin, 100 µM amsacrine, 1 µM mitoxantrone). The purine ladder was obtained after formic acid reaction. Top2, no drug treatment; Control, no top2, no drug treatment. Numbers correspond to genomic positions of the nucleotide covalently linked to top2. YWT, yeast wild-type enzyme; yS740W, top2S740W. Double-headed arrows correspond to DNA cleavage sites with a 4-base pair stagger and represent potential DNA double-strand breaks.

Calcium-promoted DNA Cleavage Sites Differ between the Top2S740W and the Wild-type Enzyme-- To investigate whether the altered DNA cleavage activity of the top2S740W was drug-dependent, we compared the calcium-promoted DNA cleavage (29) for the Ser740 right-arrow 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 Ca2+; 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 5'-overhang, as expected for concerted top2-induced double-strand cleavage (2, 5, 7). These results suggest that the top2S740W mutation also alters DNA cleavage in the absence of a topoisomerase II poison.


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Fig. 2.   Ser740 right-arrow 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 32P at the 5'-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 MgCl2 or 5 mM CaCl2 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, top2S740W. Double-headed arrows correspond to DNA cleavage sites with a 4-base pair stagger which represent potential DNA double-strand breaks.

Altered Base Preference of the Etoposide-stabilized Cleavage Complexes for the Mutant Top2S740W-- As described above, the top2S740W 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 C-1 (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 G+5. Top2S740W also demonstrated a strong preference for C-1. In addition, a novel preference for C at position -2 (94 out of 176 sites) in combination with the complementary G at position +6 (84 out of 176 sites) was also seen. To focus on the impact of the Ser740right-arrow Trp mutation on base preference, we analyzed separately those DNA cleavage sites exclusively detectable for top2S740W or for the wild-type enzyme (Fig. 3B). These unique DNA cleavage sites did not show any clear C-1 or G+5 preference for either protein, which is expected because both proteins have a C-1/G+5 preference. However, top2S740W still showed a significant preference of C at position -2 (45 out of 63 sites) in combination with a preference for the complementary G at position +6 (39 out of 63 sites). In addition, a chi-square test indicated that the combination of the C-1 and C-2 preference in the top2S740W was not significantly more frequent than having C-1 or C-2 alone. Thus, the novel C-2 base preference in the top2S740W is independent of the C-1 preference. In contrast, the cleavage sites unique for the wild-type protein tended to exclude sites with C+2 (7 out of 54 sites) and with G+6 (9 out of 54 sites). These data show a change in the protein-DNA interaction resulting from the Ser740right-arrow Trp mutation, leading to an extension of the base preference for the C-2 position in the presence of etoposide.


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Fig. 3.   Probability of the observed base frequency deviations at top2 cleavage sites for the wild-type enzyme or for the Ser740 right-arrow Trp mutant top2 in the presence of 100 µM etoposide. 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 < 0.001) greater or lower than expected.

Enhanced Salt and Heat Stability of the Cleavage Complexes Mediated by Top2S740W in the Presence of Etoposide-- The effect of the Ser740right-arrow Trp mutation on the stability of specific top2·DNA 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 Ser740 right-arrow 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 etoposide-stabilized cleavage sites were readily reversible for the wild-type protein. In contrast, a number of cleavage sites induced by top2S740W 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 top2S740W was also observed in the other c-myc DNA fragments (data not shown).


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Fig. 4.   Cleavage complexes stabilized by 100 µM etoposide with top2S740W exhibit enhanced heat stability. The DNA fragments were the same as described in Fig. 2. 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 100 µM etoposide. The reactions were then incubated at 65 °C for the indicated times before the addition of SDS and proteinase K. Top2, no drug treatment; Control, no top2, no drug treatment. Numbers correspond to genomic positions of the nucleotide covalently linked to top2. yWT, yeast wild-type top2; yS740W, top2S740W. Double-headed arrows correspond to DNA cleavage sites with a 4-base pair stagger and represent potential DNA double-strand breaks.

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 top2S740W and the wild-type enzyme (Fig. 5A). By contrast, all etoposide-stabilized cleavages induced by top2S740W 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 top2·DNA complexes formed with the top2S740W enzyme.


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Fig. 5.   Cleavage complexes stabilized by 100 µM etoposide with top2S740W 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 µM 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, top2S740W.

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 top2S740W 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 top2S740W 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 top2S740W protein.


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Fig. 6.   Cleavage complexes induced by top2S740W 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 µl 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 1× 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, top2S740W.

Reversibility of the Etoposide-induced Cleavage Complexes Is Base Sequence-dependent-- Because the top2S740W 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 top2S740W (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 C-1 (78 out of 89 sites) in combination with a less strong C-2 preference (54 out of 89 sites) (Fig. 7B). Complementary preference was observed on the opposite strand with preference (although weaker) for G+5 (53 out of 89 sites) and G+6 (36 out of 89 sites) as well. In contrast, rapidly reversible cleavage sites did not show any base preference at positions -1 and -2. However, they displayed a significant G+5 and G+6 base preference, indicating DNA cleavages in the complementary strand with a C-1 and C-2 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.


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Fig. 7.   Probability of the observed base frequency deviations at top2S740W cleavage sites showing fast or slow reversibility. Reactions were performed in the presence of 100 µM etoposide. Position 0 corresponds to the cleavage site. Panel A, 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. Panel B reflects the base distribution at each position. Underlined numbers represent base frequencies significantly (p < 0.001) greater or lower than expected.


    DISCUSSION

A number of factors contribute to the sensitivity of cells toward agents targeted to the type II topoisomerases (32-35). 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 sensitivity have not been defined. Decreased drug binding to the top2·DNA complex has been reported for quinolone-resistant DNA gyrase with a Ser83 right-arrow Trp mutation in the A subunit (7, 38). A homologous mutation in the yeast top2 gene, which changes Ser740 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 Ser740 right-arrow Trp mutation in yeast top2 affects the DNA-protein interactions. In the absence of any drug, the calcium-promoted DNA cleavage sites of top2S740W 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 alpha 4 DNA-recognition 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 Ser740 on the protein surface and its close proximity to the DNA. In addition, Ser740 is approximately 3.0 Å from Tyr734, and these residues could form a hydrogen bond. Thus, the Ser740right-arrow Trp mutation could alter the DNA-top2 interaction directly by intercalation as well as indirectly by changing the enzyme conformation and modifying DNA recognition.


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Fig. 8.   Position of the Ser740 right-arrow Trp mutation in the yeast top2 and hypothetical DNA interactions. Drawings were generated using the program Quanta (Version 97) based on coordinates reported in Ref. 17. Panel A, ribbon representation of the crystal structure of the 92-kDa fragment of the yeast enzyme (17) with a DNA fragment modeled into each of the putative DNA binding sites (18). The positions of the Ser740 in each top2 subunit are shown as van der Waals spheres and are highlighted by green arrows. Panel B is a close view of the DNA binding region, presenting the proximity of the Ser740 mutation to the DNA. Ser740 is shown in a ball-and-stick model. The numbering of amino acid residues was corrected according to Ref. 17.

The Ser740 right-arrow 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 top2S740W-induced DNA cleavages are dependent on etoposide interaction. First, calcium-promoted cleavages, although presenting an altered DNA-protein 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 drug-stacking 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 Ser740 right-arrow Trp mutation might directly or indirectly change at least overlapping quinolone-protein and etoposide-protein interaction domains. Furthermore, we found a novel C-2 base preference in the top2S740W in the presence of etoposide. This new C-2 preference proved to be statistically independent of the common C-1 preference. These findings suggest a model of a more relaxed drug binding site for the Ser740right-arrow Trp enzyme allowing etoposide to interact with C-2 in addition to C-1. This model does not conflict with the hypothesis of etoposide acting at the DNA-protein interface (5, 21, 30) because the altered interface between the top2S740W 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 top2·DNA complex.

Further structural studies with wild-type and Ser740 right-arrow 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.

    ACKNOWLEDGEMENTS

We thank Drs. Q. Liu and J. C. Wang for providing data to generate Fig. 8. We also thank Dr. T. Macdonald, Department of Chemistry, University of Virginia, Charlottesville, and Drs. P. R. McGuirk and T. D. Gootz, Pfizer, for providing topoisomerase II inhibitors.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by Deutsche Forschungsgemeinschaft Grant Str 527/1-1, Bonn, Germany.

Supported in part by Grant CA52814 from the NCI, National Institutes of Health, and the American Lebanese Syrian Associated Charities.

** To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, Bldg. 37, Rm. 5D02, NIH, Bethesda, MD 20892-4255. Tel.: 301-496-5944; Fax: 301-402-0752; E-mail: pommier{at}nih.gov.

    ABBREVIATIONS

The abbreviations used are: top2, topoisomerase II; PCR, polymerase chain reaction; HTH, helix-turn-helix.

    REFERENCES
Top
Abstract
Introduction
References
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