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Mutation P732L in Human DNA Topoisomerase II Abolishes DNA Cleavage in the Presence of Calcium and Confers Drug Resistance

Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, United Kingdom

Received June 21, 2005; accepted October 20, 2005

	Abstract

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The anti cancer drug methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) targets human DNA topoisomerase II. We report here the first selection with mAMSA of resistant human topoisomerase II. Random mutagenesis of human DNA topoisomerase II cDNA, followed by selection in yeast for resistance to mAMSA, identified P732L. This mutant was 10-fold less sensitive to mAMSA and cross-resistant to other chemotherapeutic agents such as etoposide, ellipticine, methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and doxorubicin. P732L is functional but has reduced strand passage activities and altered DNA binding compared with the wild-type protein. It has drastically altered cleavage properties compared with the wild-type enzyme. It cleaved a 40-base pair (bp) DNA substrate in the presence of magnesium but at positions different from that of the wild-type protein. More striking is that P732L was unable to cleave the 40-bp DNA substrate, a 500-bp linear substrate, or a 4.3-kilobase supercoiled substrate in the presence of calcium ions. This is the first report of a topoisomerase II mutation abolishing the ability of calcium to support DNA cleavage. This provides evidence for metal ion requirement for the phosphoryltransfer reaction of topoisomerase II and a possible mechanism for drug resistance.

Humans have two topo II isoforms, termed and . We have shown that both human topo II isoforms are poisoned by the anticancer drug mAMSA both in vitro and in vivo and that they had similar DNA cleavage site determinants in the presence of this cytotoxic drug (Marsh et al., 1996; Meczes et al., 1997). mAMSA increases the formation of DNA cleavage sites by inhibiting the religation reaction (Robinson et al., 1991). Further evidence that mAMSA can target both isoforms has come from studies with a pair of murine embryo fibroblast cell lines with and without topo II (Errington et al., 1999). In another study, in which topo II was transfected back into cells lacking topo II, restoration of sensitivity to amsacrine (mAMSA) was seen, confirming topo II as a target of mAMSA (Dereuddre et al., 1997).

All topo II poisons stabilize the normally transient cleavage intermediates or cleavable complexes; this DNA damage triggers apoptosis (Tewey et al., 1984; Pommier et al., 1991). Mutations in topo II genes that impair the ability of the drug to bind to the enzyme or decrease the DNA binding and cleavage properties of topo II enzymes can cause drug resistance (Pommier, 1993).

Several model systems have been used to select mAMSA drug resistance mutations in topo II to elucidate how this drug interacts with its target. Two mutations in the genes encoding bacteriophage T4 topo II have been reported to give rise to mAMSA-resistant T4 (Freudenreich et al., 1998). The yeast model system uses a strain bearing a temperature-sensitive topo II that can be rescued at the nonpermissive temperature by a plasmid-borne functional topo II. This system has been used to select mAMSA-resistant mutations in both yeast topo II and human topo II. Mutations at two positions in yeast topo II were identified, K478A (within the conserved PLRGK motif) and A642G (Wasserman and Wang, 1994). Use of this system also identified two mutations in human topo II, R486K and E571K, that conferred resistance to mAMSA. Arg486 is in the conserved PLRGK motif (Patel et al., 2000). (The bold, italic letter shows the position of the mutated amino acid residue.) In addition, several human cell lines that are resistant to mAMSA have been shown to bear a point mutation R486K in human topoisomerase II (Bakic et al., 1986; Hinds et al., 1991; Lee et al., 1992; Kubo et al., 1996)

In this article, we report the first use of the yeast model system to select for mAMSA resistance in human topo II. Randomly mutagenized plasmids encoding human topo II were transformed into a Saccharomyces cerevisiae strain and grown in the presence of mAMSA. htopo II bearing a P732L mutation was selected. In vitro characterization of P732L revealed that this mutation had distinct cleavage properties compared with the wild type. To our knowledge, it is the first mutation in a type II topoisomerase that is able to cleave DNA in the presence of magnesium ions but not calcium ions.

	Materials and Methods

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Reagents
Chemicals and Drugs. mAMSA was obtained from the National Cancer Institute (Bethesda, MD). AMCA and mAMCA were supplied by Prof. B. C. Baguley (Auckland Cancer Society Research Center, University of Auckland, Auckland, New Zealand). Etoposide, mitoxantrone, doxorubicin, and ellipticine were purchased from Sigma (St. Louis, MO). DNA sequencing was carried out by Lark Technologies Inc (Essex, UK). Oligonucleotides were ordered from Invitrogen (Paisley, UK). Radiochemicals were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK) and enzymes from New England Biolabs (Hertfordshire, UK) or Promega (Southampton, UK). kDNA was purchased from TopoGEN, Inc. (Port Orange, FL).

Yeast Strains and Expression Plasmids.

• JN394t2–4: ISE2 urs3–52 top2-4 rad52::LEU2. A yeast strain carrying a temperature sensitive mutation in the yeast topoisomerase II gene that makes yeast viable at 25°C but nonviable at 35°C (Nitiss, 1994).
  
• JEL1top1: A protease-deficient S. cerevisiae strain for the expression of topo II: leu2 trp1 ura3–52 prb1–1122pep4–3 his3::PGAL4.
  
• YEphTOP2KLM: carries the first five codons of the TOP2 gene fused to codons 46 to 1621 of the human TOP2 cDNA (wt) (Meczes et al., 1997).
  

Hydroxylamine Mutagenesis and Selection in the Presence of mAMSA. Random mutagenesis and selection for drug resistance was as described previously (Leontiou et al., 2004), except that the selecting agent was mAMSA at 190 µM (74.7 µg/ml), twenty times the IC₅₀ value in yeast (Meczes et al., 1997). The wild-type htopo II (wt) cDNA was mutagenized by exposure to hydroxylamine. This library of randomly mutated plasmids was then transformed into the yeast strain JN394 top2-4 in which growth at the nonpermissive temperature is dependent upon a functional plasmid-borne topo II. Drug-resistant transformants were selected by exposure to 190 µM (75 µg/ml) mAMSA in liquid culture for 96 h. The surviving yeast were plated on drug-free plates and grown for 5 to 7 days at 35°C; approximately 500 colonies grew. Of these, 36 were resistant when re-streaked onto plates containing mAMSA. Plasmids were rescued from these resistant yeast transformants. Yeast strain JN394 top2-4 was retransformed with these plasmids, and the transformants were grown on plates containing mAMSA to confirm whether their drug resistance was plasmid-borne. After retransformation, only three of the 36 grew on plates containing 10 µg/ml mAMSA, so only these three had plasmid-borne resistance. To confirm that a single point mutation in YEphTOP2KLM was the cause of the drug resistance phenotype, a fragment exchange was performed (Leontiou et al., 2004). Fragment exchange of the 1674-bp region that contained the C-to-T change, resulting in the P732L mutation into an un-mutagenized vector, was performed. The fragment-exchanged construct was transformed into the JN394 top2-4 strain. The temperature sensitive mutation in the endogenous topo II gene in this strain permits growth of the yeast cells at 25°C but not 35°C.

Drug sensitivity was determined via the minimum lethal concentration (MLC), after short-term exposure to mAMSA in liquid culture or by continuous drug exposure by plating the yeast on agar plates containing drugs at a range of concentrations and incubation at 35°C for 3 to 5 days (Leontiou et al., 2004).

Protein Expression and Purification
Recombinant htopo II proteins were expressed in the yeast strain JEL1top1 bearing plasmid YEphTOP2KLM or YEphTOP2P732L. Recombinant proteins were produced as described previously (Austin et al., 1995).

Strand Passage Assays
Decatenation and relaxation assays were carried out in relaxation buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 mM dithiothreitol, 100 mM KCl, and 30 µg/ml bovine serum albumin) with 1 µg of supercoiled pBR322 plasmid DNA or 400 ng of kinetoplast DNA, 2 mM ATP, and 10 mM MgCl₂, as described previously (Austin et al., 1995).

DNA Cleavage Assays
The cleavage properties of wt and P732L proteins were tested by incubating the protein with a ³²P-end labeled 40-bp linear DNA substrate. The cleavage religation equilibria of wt and P732L were tested in the presence of 10 mM MgCl₂ or CaCl₂ to test the non–drug-stimulated cleavage properties of the enzymes and in the presence of mAMSA (10 µg/ml) to compare the drug-stimulated cleavage of wt and P732L (Osheroff and Zechiedrich, 1987). To calculate the relative cleavage under these conditions, the amount of end-labeled cleaved product in several experiments was quantified using filmless autoradiographic analysis using a Fujifilm BAS-1500 machine. The amount of cleavage with wild-type protein in the presence of mAMSA and magnesium was taken as 100%, and the cleavage under different conditions was calculated relative to this (Marsh et al., 1996).

Cleavage was also analyzed on a 564-bp end-labeled fragment, on 4.3-kilobase linearized pBR322, and on supercoiled plasmid DNA. In all of the cleavage experiments, the protein was in excess over the DNA. For example, in the 40-bp cleavage experiments, there was a 10-fold molar excess of protein over DNA (3 and 0.3 pmol, respectively), whereas in the supercoiled cleavage, there was only a 2-fold molar excess of protein over DNA (1 and 0.5 pmol, respectively).

Reversal of Cleavage Assays
A 564-bp fragment of pBR322 was amplified by polymerase chain reaction, and reversibility experiments were carried out as described in (Leontiou et al., 2004).

Surface Plasmon Resonance
The DNA binding properties of the wt and P732L proteins were tested by surface plasmon resonance on a BIAcore 2000 (BIAcore AB, Uppsala, Sweden). The DNA substrates used were a 40-bp linear oligonucleotide (the same substrate used for the cleavage assays), a synthetic four-way junction substrate, and a piece of bent DNA from the Drosophila melanogaster genome. The linear 40-bp substrate contains a single mAMSA cleavage site and allows the binding of one topo II dimer. The 4wj substrate contains the sequence of the 40-bp linear substrate along two adjacent arms, with the cleavage site straddling the point of strand exchange. The bent DNA substrate is a 189-bp AT-rich region from the D. melanogaster genome that forms a bend.

Topo II-DNA binding was carried out in 50 mM Tris-HCl, pH 7.7, 1 mM EDTA, 1 mM EGTA, 100 mM KCl, 1 mM ATP, 0.05% (v/v) Tween p20, and 1% (v/v) Triton X-100. Protein samples were injected using the BIAcore function `kinject' at a flow rate of 70 µl/min. The protein was injected for 60 s, and the dissociation phase was recorded for 180 s at 25°C. After injection, the chip was regenerated by a 30-s pulse of 0.5% (w/v) SDS followed by a 60-s injection of 0.5 M NaCl. The concentration range of protein used was 5 to 60 nM. When protein becomes bound to DNA on the sensor chip, the refractive index of the medium at the chip surface is affected. This alteration is referred to as a change in RU, which represents an indirect measure of the amount of bound protein.

The association and dissociation data were modeled and analyzed by using BIAevaluation software, version 3.0 (BIAcore). The association and dissociation phases were analyzed for several protein concentrations. Data were fitted by using the numerical integration method. The residual plots and the ² values were used to assess the appropriateness of the various models available for analyzing the sensor data. In general, values of ² < 10 were accepted.

Topo II DNA binding data were produced for various protein concentrations and were fitted simultaneously, assuming the 1:1 Langmuir model. The Langmuir model assumes that the analyte is both monovalent and homogeneous, that the ligand is also homogeneous, and that binding events are independent. The binding of topo II to DNA follows first-order kinetics: A + B AB. The fitting parameters provide estimates of both association and dissociation rate constants (k_a and k_d), and from these values, the equilibrium constants K_A and K_D were calculated (K_D = k_d/k_a and K_A = k_a/k_d) (Leontiou et al., 2003).

Protein Structure Analysis. Protein structures were manipulated and displayed using QUANTA, release 4.1.1. (Accelrys, San Diego, CA) on an SGI workstation (Mountain View, CA). Human DNA topoisomerase II was modeled on the yeast crystal structure using the program Modeler (Sali and Blundell, 1993), as described by West et al. (2000). The structure was used to model the effect of the proline-to-leucine mutation at position 732. The model was subjected to an energy minimization cycle using Charmm (50 steps steepest descents). A new structure was created in which the Pro-to Leu mutation was created by the edit command of Quanta. This file was subjected to the identical energy minimization procedure as before. The two files (energy-minimized wild type and energy-minimized P732L were superimposed, and structural differences were investigated). Visual inspection revealed small, localized changes, and a full root-mean-square analysis of the structural differences was not considered necessary.

	Results

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Selection and in Vivo Characterization of mAMSA Resistant Yeast Transformants
To identify mutations in DNA topo II that confer resistance to mAMSA, a forced molecular evolution approach was used. Three plasmids conferred resistance to mAMSA. Two of the plasmids (mAMSA 1 and 13) that supported growth on 10 µg/ml bore the same point change, a C-to-T change at base 1394, resulting in a glycine-to-aspartic acid substitution at amino acid residue 465 (G465D). One transformant, mAMSA 7, was also able to grow on plates containing 200 µg/ml. This most resistant transformant, mAMSA 7, contained the plasmid YEphTOP2KLM, bearing a transition mutation C T at position 2195 bp, resulting in a proline-to-leucine amino acid change at residue 732, YEphTOP2P732L. Here we report the detailed characterization of P732L.

P732L Is Resistant to All Antitopoisomerase II Drugs Tested
P732L was able to complement the endogenous yeast enzyme at 35°C, confirming that this mutated protein is functional in vivo. The level of drug resistance to the selecting agent mAMSA was assessed in 2 ways. It was quantified using the MLC method (Nitiss, 1994). JN394 top2-4 were transformed with plasmid encoding wt or P732L and were grown in the presence of different concentrations of mAMSA for 6 and 24 h (Fig. 1, A and B). Figure 1 shows the percentage relative survival versus drug concentration; the drug concentration that gave 100% survival was taken as the MLC. The MLC for wt transformants on mAMSA was 1.5 µg/ml after 6 h and 2.4 µg/ml after 24 h (Fig. 1, A and B). Yeast transformants expressing P732L survived at higher mAMSA levels; thus, these transformants were exposed to a higher dose range. The MLC for P732L transformant on mAMSA was 14.8 µg/ml after 6 h and 24.8 µg/ml after 24 h (Fig. 1, A and B). Thus, P732L conferred 9.9-fold resistance to mAMSA compared with the wild type. This difference was found to be statistically significant when a paired, two-tailed t test was employed (p < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Effects of mAMSA on the viability of yeast strain JN394 top2-4 transformed with plasmids encoding recombinant wt or P732L at 35°C. Viable counts were determined after growth of yeast transformants in the absence or presence of mAMSA at the concentrations indicated for 0, 6, or 24 h. The values are expressed as a percentage of viable counts measured for time 0, percentage of relative survival. The results are the average of three independent experiments. Error bars represent the S.E.M. of three experiments. A shows the 6-h exposure to mAMSA and B shows the 24-h exposure. Plots of JN394 top2-4-expressing wt () and JN394 top2-4-expressing P732L () are shown.

Cross-resistance to other drugs was tested qualitatively by continuous exposure to drug (Leontiou et al., 2004). JN394 top2-4 expressing either wt or P732L was grown on YPDA plates containing different drugs, including mAMSA, AMCA, mAMCA, etoposide, ellipticine, or doxorubicin. P732L was found to be resistant to all the drugs tested, ranging between 2- and 50-fold (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 The drug sensitivity profile of JN394 top2-4 expressing wt or P732L in the presence of different cytotoxics at 35°C The yeast strain JN394 top2-4 was transformed with recombinant plasmid encoding either wt or P732L. The transformed strains were tested for drug sensitivity to different concentrations of topo II poisons. Equal amounts of each transformant as determined by optical density were plated in a series of dilutions onto adenie-supplemented yeast extract/peptone/dextrose with drug and were grown at 35°C for 5 days. After 5 days, the extent of growth was assessed by comparing the number of yeast colonies with the wild type. The result shown here is the amount of growth of yeast colonies. Comparisons of the level of growth from several dilutions on plates containing several different drug concentrations allowed approximate -fold resistance values to be determined. The experiment was performed in duplicate.

P732L Shows Decreased Strand Passage Activities Compared with the wt Protein
Wild-type and mutated proteins were expressed in yeast, and the enzymes were purified to >95% homogeneity (Fig. 2A). Purified P732L and wt proteins were assayed for strand passage activity by both decatenation of kDNA and relaxation of supercoiled plasmid DNA. P732L had significantly reduced relaxation activity (56.5 ± 8.2%) compared with the wild-type protein and reduced in vitro decatenation activity (74.4 ± 29.3%) compared with the wild-type protein (Fig. 2B).

View larger version (31K): [in this window] [in a new window]   Fig. 2. A, a 7.5% SDS-PAGE gel loaded with 1.5 µg of wt or P732L and stained with Coomassie blue. B, strand passage activity, histograms of the relative relaxation activity () and relative decatenation () for wt and P732L. wt is set to 100%. Means were calculated from at least three experiments; error bars represent 1 S.D. from the mean. C, effect of Mg2+ concentration on decatenation by wt () and P732L (). Mean and S.D. are derived from at least three experiments. D, effect of ATP concentration by wt () and P732L (). Mean and S.D. are are derived from at least three experiments. E, DNA binding of human topo II wt and P732L by surface plasmon resonance to four-way junction DNA. The experiment was carried out at 25°C, the association phase was 80 s. Resonance units are plotted versus time.

P732L Has Altered DNA Binding Properties Compared with wt
wt bound to all three substrates tested (linear, four-way junction, and bent DNA) with comparable affinity, the K_D values were in the range of 1 to 3 nM (Leontiou et al., 2003). For the wild-type protein, the data best fit the simple 1:1 (Langmuir) model for all three substrates. P732L bound to four-way junction DNA with similar kinetics to wt, with a K_D of 1 nM (Fig. 2E). However, unlike the wt, P732L binding to the linear and bent DNA substrates did not fit a Langmuir binding model, so no K_D values could be determined for these substrates.

P732L Protein Has Altered DNA Cleavage Properties Compared with wt
Cleavage of a 40-bp Oligonucleotide. The ability of the wt and P732L proteins to cleave a 40-bp linear DNA substrate was tested (Fig. 3A). This substrate contains one mAMSA site that is cleaved strongly in the presence of mAMSA and weakly in its absence, to produce two cleavage products of 21 and 15 nucleotides (Marsh et al., 1996).

View larger version (50K): [in this window] [in a new window]   Fig. 3. A, cleavage of the 32P-labeled 40-bp DNA substrate by wt and P732L. Cleavage was carried out in the presence of no protein (lane 1) or 1.2 µg of wt (lanes 2–5) or P732L (lanes 6–9), plus Mg2+ (lanes 2 and 6), Mg2+ and mAMSA (lanes 3 and 7), Ca2+ (lanes 4 and 8), and Ca2+ and mAMSA (lanes 5 and 9). Cleavage products were quantified by filmless autoradiographic analysis using a Fujifilm BAS-1500 machine. This figure shows an individual experiment that is typical of at least three others. B, cleavage of the 32P-labeled 40-bp DNA substrate by wt and P732L. Cleavage was carried out in the presence of no protein (lane 1) or 1.2 µg of wt (lanes 2 and 3) or P732L (lanes 4 and 5), plus Mg2+ (lanes 2 and 4) or Mg2+ and etoposide (lanes 3 and 5). Cleavage products were quantified by filmless autoradiographic analysis using a Fujifilm BAS-1500 machine. C, cleavage of the 32P-labeled 564-bp DNA substrate by wt and P732L. Protein (1.2 µg) was incubated with the 564-bp 32P-labeled pBR322 substrate for 30 min at 37°C in the presence of various cations and mAMSA and either wt (lanes 2–5) or 732L (lanes 6–9). Lane 1, no protein; lanes 2 and 6, Mg2+ + mAMSA; lanes 3 and 7, Ca2+ + mAMSA; lanes 4 and 8, Mn2+ + mAMSA; lanes 5 and 9, Ni2+ + mAMSA.

Cleavage of the 40-bp substrate by P732L was very different from cleavage of this substrate by wt protein, in the presence of Mg²⁺ ions, it produced a ladder of approximately 15 cleavage products (Fig. 3A, lane 6), not the expected two products of 15 and 21 nucleotides. The amount of cleavage by P732L with magnesium was greater than wt under the same conditions (Fig. 3A, lanes 2 and 6). This cleavage by P732L was not further stimulated by mAMSA (Fig. 3A, lane 7), unlike wt, the magnesium cleavage of which was stimulated 13-fold by mAMSA. It is striking that P732L was not able to cleave DNA in the presence of calcium ions (Fig. 3A, lane 8), even when mAMSA was added in the reaction (Fig. 3A lane 9). Cleavage of this 40-bp oligonucleotide by wt can also be stimulated by etoposide. However, etoposide did not further stimulate cleavage by P732L on this 40-bp substrate, the cleavage products gave the same pattern and intensity with or without drug (Fig. 3B), indicating that P732L cleavage of the 40-bp oligonucleotide substrate in the presence of magnesium is not further stimulated by mAMSA or etoposide.

Cleavage of a 564-bp Fragment of pBR322. To further investigate the cleavage characteristics of P732L, a 564-bp fragment of pBR322 was isolated by polymerase chain reaction and end-labeled. P732L did not produce the same-size cleavage products as the wt protein. Cleavage of the 564-bp substrate was carried out in the presence of magnesium and mAMSA (Fig. 3C, lanes 2 and 6). Under these conditions, the P732L protein (Fig. 3C, lane 6), produced different cleavage products compared with wt with magnesium and mAMSA (Fig. 3C, lane 2). Cleavage of the 564-bp substrate by P732L was also carried out in the presence of calcium ions and mAMSA; P732L was unable to support cleavage in the presence of calcium ions (Fig. 3C, lane 7), unlike wt under these conditions (Fig. 3C, lane 3).

Cleavage with manganese and nickel was analyzed for wt and P732L (Fig. 3C, lanes 4, 5, 8, and 9). Manganese and nickel supported cleavage with both wt and P732L. The P732L cleavage with manganese or nickel was more promiscuous than wt, as seen with magnesium, cleaving in more positions resulting in a different cleavage pattern. wt cleavage with nickel was less intense than with manganese; this difference was not seen with P732L.

P732L Forms More Stable Cleavable Complexes in the Absence or Presence of mAMSA Compared with wt
The salt reversal of the complexes on the 564-bp substrate was assessed to determine the in vitro stability of the complexes with either wt or P732L (Fig. 4). The level of cleavage with wt with only magnesium was too low to accurately determine the reversal times, but salt reversal of wt with magnesium and mAMSA is shown in Fig. 4A, the half-lives of complexes were 5 min, and all the complexes had reversed within 20 min. In contrast, P732L complexes in the presence of magnesium either with or without mAMSA (Fig. 4, B and C, respectively) were all still present after 20 min, showing that their stability was greater than wt and this stability was not drug-dependent.

View larger version (104K): [in this window] [in a new window]   Fig. 4. An autoradiograph of a 564-bp 32P-labeled pBR322 DNA substrate after incubation with or without mAMSA, in the presence of 10 nM MgCl2 and the indicated protein. Reactions were terminated at the noted time points (0–20 min).

View larger version (41K): [in this window] [in a new window]   Fig. 5. A, cleavage of the 32P-labeled 4.3-kb pBR322 probe in the presence of mAMSA or etoposide. Lane 1, no protein; lanes 2 and 4, wt; lanes 3 and 5, P732L; lanes 2 and 3, with mAMSA; lanes 4 and 5, with etopooside. B, cleavage of supercoiled plasmid DNA by wt and P732L. Protein (4 µg) was incubated with 1.5 µg of supercoiled plasmid DNA in relaxation buffer with 10 mM MgCl2 or CaCl2± mAMSA with either wt (lanes 2–5) or P732L (lanes 6–9) for 30 min at 37°C. Control supercoiled plasmid (lane 1). Lanes 2 and 6, Mg2+; lanes 3 and 7, Mg2+ + mAMSA; lanes 4 and 8, Ca2+; lanes 5 and 10, Ca2+ + mAMSA.

Cleavage of Supercoiled Plasmid Substrate by wt and P732L in the Presence of Mg²⁺ or Ca²⁺. To test whether P732L was able to cleave longer substrates in the presence of calcium, we tested the magnesium and calcium cleavage of supercoiled plasmid DNA both in the presence and in the absence of mAMSA (Fig. 5B). wt cleaved weakly with magnesium (Fig. 5B, lane 2) and well with calcium (Fig. 5B, lane 3), and with either magnesium or calcium, mAMSA increases the level of cleavage (Fig. 5B, lanes 4 and 5). P732L showed more cleavage than wt with magnesium alone (Fig. 5B, lanes 6 and 2, respectively). No cleavage was seen with P732L and calcium (Fig. 5B, lanes 8 and 9), in agreement with the experiments on the 40- and 564-bp linear substrates.

Modeling of the Central Breakage Domains of Wild-Type topo II and P732L. Type II topoisomerases have three structural domains: an N-terminal ATPase, a central breakage-rejoining domain composed of the A' and B' subfragments, and a C-terminal domain (Austin et al., 1995). Residue 732 lies in the A' subfragment, which spans residues 722 to 1200 in human topo II. The A' subfragment can be further subdivided into three sections. The first section of A' spans residues 722–858 and is termed the "CAP-like" domain as it contains a fold similar to one found in the catabolite activator protein and histone H5 (Berger et al., 1996). This portion of the A' subfragment also contains the active site tyrosine (Tyr820) responsible for forming the phospho-tyrosine linkage with DNA. Pro732 lies at the end of the A' 1 helix within the CAP-like fold. An alignment of the "CAP-like" domain from human topo II, human topo II, yeast topo II, and Escherichia coli gyrase A (Fig. 6a) shows that this region is conserved between species. Pro732 is a highly conserved residue; a proline is found at the position equivalent to 732 in type II topoisomerases from bacteria to humans.

View larger version (42K): [in this window] [in a new window]   Fig. 6. A, alignment of the "CAP"-like region of human topo II () compared with that from human topo II (), yeast topo II (Y), and DNA gyrase A (A). The proline equivalent to Pro732 is shown in light blue, the arginine equivalent to Arg729 is shown in green, and the active site tyrosine is shown in yellow. B, a diagram of one monomer of human topo II showing the location of the P732L mutation in pale blue, the active site tyrosine in yellow, and Arg729 in light green. The location of acidic residues involved in metal ion co-ordination is also shaded. The EGDSA motif containing Glu477 is shown in dark green and the IMTDQD(Q/H)DGSH loop, containing Asp557, Asp559, and Asp561 is shown in red. C, schematic adapted from a model previously proposed by Noble and Maxwell (2002). This shows a two-metal-ion mechanism for the phosphoryl transfer reaction involved in DNA cleavage by DNA topo II. The coloring is equivalent to that seen in A and B. D, a stereo image of the region immediately surrounding residue 732; the wild-type sequence is shown in blue. The mutant chain is green with the leucine side chain shown in red, above which the wild-type proline ring can be seen. The residue numbering is that of human topoisomerase II -1, which is a 1621-residue protein, not human topoisomerase II -2, which is a 1626-residue protein (Q02880 [GenBank] ).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Mutation P732L confers in vivo drug resistance of 10-fold to mAMSA in an MLC assay and was cross-resistant to all the topo II drugs tested, including AMCA, mAMCA, etoposide, ellipticine, and doxorubicin (Fig. 1 and Table 1). P732L is functional in vivo, as our selection screen is dependent upon complementation in yeast. Although P732L was functional in vivo, its in vitro strand passage activity is reduced compared with the wild type. Binding of P732L to linear DNA substrates was altered, but binding to a four-way junction substrate was similar to the wild-type enzyme (Fig. 2E).

Our in vitro experimental data with P732L protein indicates that it has altered cleavage properties. Most striking is that calcium could not support cleavage by P732L, unlike wt. Cleavage by P732L of a 40- or 564-bp linear substrate showed the same differences: greater cleavage with magnesium alone, no drug enhancement, and no cleavage with calcium. On the 40- and 564-bp substrates, cleavage occurred at more sites compared with the wt enzyme (Fig. 3, A–C, and Fig. 4). However, on the 4.3-kb linearized pBR322, the cleavage pattern appeared similar to wt (Fig. 5A). This suggests that the altered DNA binding seen on 40- and 189-bp bent DNA substrates may be affecting cleavage site specificity on the shorter DNA substrates. Cleavage reactions using supercoiled plasmid substrate confirmed that calcium did not support cleavage by P732L on the full-length plasmid, but magnesium did. Using the 564-bp substrate, the cleavage products generated by P732L were shown to be more stable than those with wt. This suggests an alteration of the cleavage-religation equilibrium of P732L, favoring cleavage over religation, producing longer-lived cleavable complexes compared with wt. This could account for the increase in cleavage products with magnesium in the absence of drug.

Why does this proline-to-leucine change generate a protein whose DNA cleavage reaction cannot be supported by calcium and has an altered cleavage-religation equilibria? To address this question, the position of Pro732 within the protein was analyzed by modeling this region onto the yeast structure. Modeling suggested that the leucine change caused the local peptide backbone in P732L to shift position compared with wt. Topoisomerase, however, is a very dynamic protein, and the removal of a highly conserved proline may alter the local mobility during catalysis. This cannot be accurately modeled using the currently available crystal structures of parts of the yeast protein.

A number of residues in this region have previously been demonstrated to be important for enzyme catalysis. Site-directed mutations have been made in this region of the yeast topoisomerase II (Liu and Wang, 1998). Alanine substitutions at yeast Arg690, Asp697, Lys700, Arg704, or Arg781 abolished in vivo function in a complementation assay. The equivalent residues in htopo II are Arg729, Asp736, Lys739, Arg743, and Arg820, four of which are very close to Pro732.

In the yeast protein, mutating Arg690 to alanine completely abolished in vitro cleavage activity (Liu and Wang, 1998). Yeast arginine 690 is equivalent to Arg32 in Gyrase A. Noble and Maxwell (2002) suggest that this arginine is actively involved in the phosphoryl transfer mechanism that enables topo II to cleave the DNA. The equivalent residue in htopo II is Arg729, which is only three residues away from Pro732, so local conformational changes in the P732L protein may affect the proposed role of this arginine in DNA cleavage.

Phosphoryltransfer reactions have been proposed to involve two metal ions in their mechanism (Beese and Steitz, 1991). The proposed metal binding sites have magnesium ions coordinated by acidic amino acid residues such as aspartic acid and glutamic acid (West et al., 2000). We proposed two potential metal ion binding sites in htopo II coordinated to the aspartic acids found in the IMTDQD(Q/H)DGSH loop, Asp557, Asp559 and Asp561 (West et al., 2000). A similar acidic triad is also found in topoisomerase I, and mutation of these residues reduces the number of magnesium ions bound per protein molecule (Lima et al., 1994; Zhu and Tse-Dinh, 2000). Noble and Maxwell (2002) have published a model for the phosphoryl transfer in DNA cleavage by DNA gyrase. In Fig. 6C, we have adapted the Noble and Maxwell schematic diagram to represent human topo II. Arg729 is shown in light green in Fig. 6C and illustrates the proximity of residue 732 to the hypothesized location of the DNA, active site tyrosine, and the two metal binding sites hypothesized to catalyze the DNA cleavage reaction. The predicted local conformational changes may account for the altered DNA binding to linear DNA observed by SPR. It also suggests an explanation for the cleavage in the presence of magnesium but not calcium ions. If the local conformational change alters the liganding geometry for the bound metal ions, such that calcium can no longer support the phosphoryl transfer reaction and the magnesium supported phosphoryl transfer reaction has an altered equilibrium favoring cleavage over religation, this could account for the greater stability of the cleavage complexes with magnesium and the lack thereof with calcium.

The presence of a contaminating nuclease would offer a simple explanation for the altered cleavage properties with magnesium, such as the extensive cleavage with magnesium alone and the lack of drug stimulation of cleavage in the presence of magnesium on short DNA substrates and lack of reversibility. This possibility is very difficult to exclude because the amounts of such a protein could be so low that polyacrylamide gel electrophoresis or matrix-assisted laser desorption ionization approaches would not be guaranteed to pick it up. However, we do not see this non–drug-stimulated cleavage activity with magnesium in wt or in four other mutated proteins expressed using the same system and purified by the same protocol. In addition, we do not see any DNA products that seem to be caused by nuclease activity in any of the agarose gels used for activity assays.

A study in HL-60 cells indicated that an intracellular calcium buffer could reduce the DNA cleavage by topoisomerase II in the presence of either etoposide or mAMSA. This reduction correlated with decreased cytotoxicity (Aoyama et al., 1998). The absence of cleavage with calcium may account for the drug resistance seen in yeast with P732L. However, whether removal of calcium-stimulated cleavage is an important mechanism of drug resistance in human tumors remains to be determined.

	Acknowledgements

 
We thank M. Bell for technical assistance and I. G. Cowell for assistance with the figures.

	Footnotes

 
This work was supported by Cancer Research UK (C.L.) and The Wellcome Trust.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.015933.

ABBREVIATIONS: topo, DNA topoisomerase; mAMSA, amsacrine [methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide]; AMCA, methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride; mAMCA, methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride; kDNA, kinetoplast DNA; htopo, human DNA topoisomerase; MLC, minimum lethal concentration; bp, base pair(s); wt, wild type.

¹ Current affiliation: ETH Zürich, Institute for Molecular Biology and Biophysics, ETH-Hoenggerberg, Zürich, Switzerland.

Address correspondence to: Professor Caroline A. Austin, Lab 3011, Institute for Cell and Molecular Bioscience, The Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK. E-mail: caroline.austin{at}ncl.ac.uk

	References

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