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Address correspondence to Reiner Strick, Dept. of Medicine, University of Chicago, 5841 S. Maryland Ave., MC2115, Chicago, IL 60637-1470. Tel.: (773) 834-1539. Fax: (773) 702-3002. E-mail: rstrick{at}medicine.bsd.uchicago.edu
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Abstract |
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Key Words: cations; chromosome structure; condensation; ion microscopy; topoisomerase II
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Introduction |
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The most abundant cations in the eukaryotic cell are Ca2+, Mg2+, Na+, and K+. These cations are fundamental for multiple cellular processes in every phase of life including cell growth and differentiation, development, cellcell interactions, morphology, motility, and apoptosis leading to cell death (for review see Boynton et al., 1982). The major Ca2+ storage sites in the cell are the ER, Golgi complex, mitochondria, secretory granules, and nuclear envelope (for review see Rottingen and Iversen, 2000). The K+ storage sites are mainly the cytosol, Golgi complex, and the nucleus (Schapiro and Grinstein, 2000). Multiple ion transmembrane pumps (ATPases) and exchangers are responsible for Ca2+, Mg2+, Na+, and K+ cellular influx and efflux to regulate the cellular cation concentrations from internal storage sites and to maintain osmolarity (for review see Scheiner-Bobis, 1998).
Cations have been implicated in the regulation of the cell cycle (for reviews see Boynton et al., 1982; Hepler, 1994). For example, increasing concentrations of Ca2+, Mg2+, and Na+ in the media of growing cells stimulated the mitotic rate almost twofold, whereas in contrast, K+ had no effect (Atkinson et al., 1983). In other studies, cellular Na+ concentration levels appeared to fluctuate throughout the cell cycle peaking in M and S phase, whereas the K+ nuclear and cytoplasmatic concentration levels remained unchanged (Cameron et al., 1979; Warley et al., 1983). Several studies have implicated a major role of Ca2+ in mitosis, correlating with nuclear envelope breakdown and entry into mitosis, microtubular breakdown at the meta- to anaphase transition, and a brief Ca2+ increase at the anaphase onset (Poenie et al., 1986), which led to activated chromosome motion (Groigno and Whitaker, 1998). The meta- to anaphase transition could be prevented with EGTA or the more specific Ca2+-chelator 1,2-bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid (BAPTA) in Ca2+-free medium (for review see Hepler, 1994).
The main cations interacting with DNA are Ca2+, Mg2, Na+, and K+. The divalent cations bind to the negatively charged phosphate residues of DNA in a stoichiometry of 1 mol Ca2+ or Mg2+ to 2 mol phosphate (e.g., Mathieson and Olayemi, 1975). Recent crystallization studies of B-DNA decamers or dodecamers in the presence of Mg2+ or Ca2+ confirmed a direct cation interaction with the major and minor grooves as well as phosphate oxygen atoms contributing to DNA stabilization and conformation (Minasov et al., 1999; Chiu and Dickerson, 2000). These crystallization studies resolved that Ca2+ has a higher affinity to DNA, inducing a greater DNA bending and thermal stabilization than Mg2+.
Several studies have shown that mono- and divalent cations are essential in maintaining higher order chromatin structure. For example, chromatin at low ionic strength and in the absence of divalent cations has an extended structure representing the 10-nm coil. The transformation to a more compact or 30-nm structure, that is described as a solenoid or arrangement of superbeads, could be induced by an increase of Na+ or Mg2+ and Ca2+ (for review see Felsenfeld and McGhee, 1986). Models of mitotic chromosome structures have been proposed for the folding of the chromatin fiber >30 nm, each based on the hierarchical organization of eukaryotic chromatin into loops and coils (Ohnuki, 1968; Rattner and Lin, 1985; Filipski et al., 1990). In the loop-scaffold model, highly ademine-thymine (AT)-rich DNA elements, named scaffold-associated regions (SARs) interact dynamically with nonhistone proteins to form loop anchorage sites (Paulson and Laemmli, 1977). SARs play a key role as cis elements of chromosome dynamics and as initiation elements for chromosome condensation (Strick and Laemmli, 1995). Nonhistone binding proteins like topoisomerase II (Topo II) and scaffold protein II (ScII) (homologue to hCAP-E, an SMC protein), have been implicated as partners in a nuclear complex (Ma et al., 1993) and also colocalize at the chromosomal axis (Lewis and Laemmli, 1982; Saitoh et al., 1994). Topo II and protein complexes, called condensins, including hCAP-C and -E and other SMC proteins, are essential for chromosome condensation, structure maintenance, and sister chromatid separation (Adachi et al., 1991; Schmiesing et al., 1998; Hirano, 1999).
There are several methodologies available to measure both free and bound cations in cells. Fluorescent indicators, like fura-2 were developed to detect free cations, especially Ca2+ (Poenie et al., 1986). However, these indicators encounter several technical problems; for example, they can bind nonspecifically to cell constituents or other cations (McCormack and Cobbold, 1991). Although X-ray crystallography can detect Ca2+ and Mg2+ on DNA oligonucleotides (Minasov et al., 1999), Na+ and K+ cannot be easily distinguished because of the interference with H2O. In addition, cryogenic temperatures and crystal packing effects occurring during X-ray crystallography may shift the ion distribution of the sample. X-ray microanalysis has been used to measure cations in the nucleus and cytoplasm during the cell cycle (Cameron et al., 1979; Warley et al., 1983). Although this technique involves lengthy exposures and needs substantial spectral corrections due to the presence of a high continuous background, this method is a valuable tool for chemical quantitation.
In contrast to X-ray microanalysis, which determines the atomic composition only at specific points within a biological sample and with no direct microscopic representation and depth information, secondary ion mass spectrometry (SIMS) (Benninghoven, 1987) measures the isotopic composition either in the form of stable or tracer isotopes with high sensitivity, high spatial resolution, and essentially no background. Previous SIMS investigations using BrdU-labeled human and polytene chromosomes demonstrated the use of tracer isotopes (Levi-Setti et al., 1997). SIMS signals provide a rapid visualization of the isotopic distribution within a sample, and SIMS sequential mapping renders in-depth analytical information for constructing 3D compositional images (SIMS tomography).
In this investigation, we show for the first time high-resolution analytical images of the cation composition of mammalian interphase and mitotic cells as well as of isolated metaphase chromosomes using the University of Chicago (Chicago, IL) scanning ion microprobe (Levi-Setti, 1988; Chabala et al. 1995). To preserve the ionic integrity of the analyzed cells and prevent the well-known occurrence of analytical artifacts due to the high diffusivity of cations in biological samples (Morgan et al., 1975), we used fast cryopreservation methods (freeze drying and freeze fracture) (Echlin, 1984), without any prefixations or washes. This study presents SIMS imaging evidence of cation redistribution between cytosol and chromosomes during the cell cycle and proves that Mg2+, Ca2+, K+, and Na+ are an integral part of mitotic chromatin. Our results indicate that Ca2+ directly binds to Topo II in the absence of DNA, supporting a regulatory role of Ca2+ in the reversible transition of a catalytically active Topo II to a structural DNA binding protein during mitosis.
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Results |
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Ca2+ directly binds and inactivates the enzymatic activity of chromosomal Topo II
It is well documented that Topo II localizes at the mitotic chromosome axes (Earnshaw et al., 1985; Saitoh and Laemmli, 1994). From our SIMS analyses, we derived a ratio of 3:1 Ca2+/Mg2+ on the chromosomal axis (Figs. 4 and 5). Therefore, we determined the Topo II enzymatic activity in vitro in the presence of different cation concentration ratios. This assay determines the Topo II relaxation activity of negatively supercoiled plasmid DNA or catenated kinetoplast DNA in the presence of Mg2+, which is essential for catalytic activity (Osheroff and Zechiedrich, 1987). Our results showed that Topo II activity was inhibited 5790% when the molar ratio of Ca2+/Mg2+ was in the range 1:13:1, respectively (Fig. 8 A). This supports the notion that Topo II may be enzymatically inactive at the metaphase chromosome axes.
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Discussion |
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Our main research interests have focused on cationDNA and cationprotein interactions in terms of their roles in higher order structure. Therefore, the chromatin association of Na+ and K+ supports important roles of these cations in both interphase and mitotic chromatin compaction, whereas Ca2+ and Mg2+ binding points to an essential function in mitotic chromatin compaction. Consequently, the depletion of Ca2+ and Mg2+ mitotic chromosomes, which resulted in partly decondensed structures, a process which is reversible (Zelenin et al., 1982; Earnshaw and Laemmli, 1983; Staron, 1985), points to key roles for Ca2+ and Mg2+ in specific cationchromatin binding and in the dynamics of chromatin condensation and decondensation. DNA condensation is a multimolecular, highly cooperative and delicately balanced process, which occurs in a rapid time span during each cell cycle. In the presence of cations, DNA condensation is determined by charge neutralization and not by binding to DNA per se as determined by calculations of electrostatic forces (for review see Bloomfield, 1998). The binding of cations specifically to the DNA phosphates results in decreasing the overall electrostatic Coulomb repulsion between free phosphates and adjacent DNA structures. For example, the DNA neutralization fraction of core histones was calculated at 57% by circular dichroism of nucleosome cores (Morgan et al., 1987, and references therein). Therefore, the remaining 43% of the DNA net negative charge must be neutralized by histone H1, the nonhistone proteins, polyamines, like spermine4+, spermidine3+, and putrescine2+, and especially Ca2+, Mg2+, Na+, and K+. As shown for DNA oligomer crystals (Minasov et al., 1999), the DNA charge neutralization of Ca2+, Mg2+, Na+, and K+ together resulted in a greater DNA radius reduction. The effect of Ca2+ and Mg2+ on DNA compaction and overwinding has been shown with supercoiled DNA (Adrian et al., 1990) as well as with helical DNA (Xu and Bremer, 1997). The potential of cation binding for chromosome condensation and maintenance can be seen with histone-free dinoflagellate chromosomes, which are exclusively compacted and stabilized by Ca2+ and Mg2+, at two different binding sites (Herzog and Soyer, 1983). For naked DNA, the maximal binding and compaction for Ca2+ and Mg2+ was found to be 0.63 cations/bp (or one every 3.17 nucleotides) (Koltover et al., 2000). From our total Ca2+ and Mg2+ chromosome concentration values, we determined that one Ca2+ binds to every 12.520 nucleotides (12 helical turns) and one Mg2+ to every 2033 nucleotides (23 helical turns). The discrepancy between naked DNA and chromatin can be explained due to the occupation of cation binding sites with Na+, K+, and chromatin binding proteins. In addition, even after incubating IM cells with 10 µM of the Ca2+ specific ionophore A23187 for 6 h, we detected no further increase in concentration levels of Ca2+ or other cations tested for binding on mitotic chromosomes (unpublished data). This finding supports the notion that metaphase chromosomes have no additional binding sites for Ca2+ or other cations. Therefore, we conclude that the detected Ca2+, Mg2+, Na+, and K+ cations together with polyamines, histones, and nonhistone proteins result in charge neutral mitotic chromosomes and represent the highest compacted state. During cation neutralization, this process may lead to modifications in local DNA structures, like bending toward the neutralized region, and thus facilitate nucleosome folding. Subsequently, the charge neutralization of chromosomes may be necessary to facilitate free chromosomal movement throughout mitosis.
In addition to the overall chromosome binding of Ca2+, Mg2+, Na+, and K+, we also detected that Ca2+ specifically binds to the chromosome axis, the location of the scaffold proteins and the highly AT-rich SARs, in a 3:1 Ca2+/Mg2+ ratio. Different DNA binding properties of Ca2+ and Mg2+ have been shown in DNA oligomer crystals where both divalent cations bound to the DNA phosphate groups over oxygen atoms; but in contrast to Mg2+, Ca2+ was specifically found in the minor groove of AT-rich DNA (Minasov et al., 1999). Recent NMR studies of DNA oligomeres in the presence of K+ demonstrated a stabilization of quadruplex DNA structures as implicated at the telomeres and centromeres (Marathias and Bolton, 2000). The K+ nucleolar enrichment we observed may reflect the centromeric regions mapping adjacent to the nucleolar organizing regions. Lewis and Laemmli (1982) proposed that Cu2+ or Ca2+ is needed for stabilization of chromosome scaffolding proteins, including Topo II (ScI) and ScII. It has previously been shown that 10% of the total nuclear Ca2+ is bound to the nonhistonescaffold protein fraction (Schibeci and Martonosi, 1980). Our direct binding experiments of Ca2+ with Topo II supports that Topo II is the main Ca2+-binding protein in the scaffold fraction (Fig. 8 B). Since we demonstrated that Ca2+ and not Cu2+ is enriched on the chromosomal axis using SIMS, we propose that Ca2+ is the most likely candidate for stabilization of the chromosomal scaffolding proteins, particularly Topo II. The finding of Lewis and Laemmli (1982) concerning the role of Cu2+ can be explained in that, in contrast to Ca2+, Cu2+ can bind multiple oxygen atoms of proteins with the result of oxidizing the peptide backbone and being reduced to Cu+ (Legler et al., 1985). The fact that Ca2+ also widened the minor groove, whereas Mg2+ contracted it (Minasov et al., 1999), may also have important implications for the structure and function of SARs at the chromosome axes as well as for axis-binding proteins like Topo II and ScII.
Topo II and ScII proteins comprise 40% of the overall proteins in the chromosome scaffold (Earnshaw and Laemmli, 1983), and are involved in chromosome structure maintenance at the chromosome axes (Boy de la Tour and Laemmli, 1988; Saitoh et al., 1994). Andreassen et al. (1997) colocalized Topo II and ScII only after prophase on the axes, supporting that the mitotic ScII may be present in two complexes, with the condensins and with Topo II. We showed that Topo II directly binds Ca2+ without binding DNA, whereas chromosomal ScII did not (Fig. 8 B), although Ca2+ chelation experiments depleted Topo II and ScII from mitotic chromosomes (Fig. 7). This result could be explained that Topo II binds to both Ca2+ and ScII and after chelation of Ca2+ both proteins are depleted due to possible strong proteinprotein binding (Lewis and Laemmli, 1982; Ma et al., 1993). We observed that over 8090% chelation of chromosome bound Ca2+ and Mg2+ resulted in partially decondensed chromosomes and, more importantly, in a 9095% loss of Topo II and ScII, but in no loss of hCAP-C and histones. These partially decondensed structures are most likely due to both the loss of compaction and neutralization by Ca2+ and Mg2+, as well as by the simultaneous depletion of Ca2+/Mg2+-bound Topo II. A complete chromosome collapse was not detected because of the remaining Na+ and K+, histones, and other nonhistone proteins, like hCAP-C as well as DNADNA interactions.
Finally, we found that the 3:1 chromosome axis ratio of Ca2+/Mg2+ fully inhibited Topo II catalytic activity in vitro (Fig. 8 A). In Ca2+ and Mg2+ competition studies by us and Osheroff and Zechiedrich (1987), results showed that an interaction between Ca2+ and Topo II led to inactivation of the catalytic activity by trapping Topo II onto DNA in a stabilized cleavage complex. It is reasonable that the recent findings of catalytically inactive Topo II on chromosomes at metaphase and especially anaphase (Shamu and Murray, 1992; Meyer et al., 1997; Bojanowski et al., 1998) may be explained by Ca2+ binding, and that the actual mechanism involving Ca2+-induced Topo II enzymatic inactivation could be due to the larger ionic radii of Ca2+ (0.99A) as compared with Mg2+ (0.66 A), thus altering the tertiary structure of Topo II and converting it to a solely structural DNA binding protein. If Topo II has two (or more) different binding sites for Ca2+ and Mg2+ like DNase A (Poulos and Price, 1972) or only one site is still unresolved. Interestingly, in the presence of Ca2+, DNase A had different DNA cleavage specificity and changes of the protein structure protecting the enzyme against proteolysis (Poulos and Price, 1972). It will be important to determine if the enzymatic activity of Topo II is inhibited after binding of Ca2+ due to the changing of protein structure.
The G2 checkpoint of mammalian cells requires Topo IIdependent decatenation of DNA duplexes before entry in mitosis (Downes et al., 1994). Topo II specific drugs, like VP16 given in G1 or S phase inhibit Topo II, resulting in undecatenated DNA and G2 arrest (Tobey et al., 1990). Ca2+ binds Topo II and DNA in a covalent complex, but leaves Topo II kinetically active (Osheroff and Zechiedrich, 1987). In contrast to VP16, a depletion of Ca2+ at the chromatin due to the redistribution during the cell cycle could result in active Topo II, religating DNA cleavages, and thus, explain the lack of DNA breaks during/after mitosis. After prophase, the cells are insensitive to Topo II inhibitors (Rowley and Kort, 1989), presumably because chromosomal Topo II is complexed with Ca2+ and DNA as stable cleavage complexes.
In conclusion, this investigation identifies the cations Ca2+, Mg2+, Na+, and K+ as essential participants in the maintenance of higher order structure in mammalian chromosomes particularly at mitosis due to their functions in (a) DNA electrostatic neutralization and chromosome condensation, (b) a direct interaction of Ca2+ with Topo II, and (c) regulation of Topo II as a structural chromosomal binding protein through cationprotein interactions with Ca2+ at the chromosomal axis. The distribution of these chromatin-binding cations and the scaffold proteins are represented in our chromosome model (Fig. 9). Thus, the cations Ca2+, Mg2+, Na+, and K+ in addition to polyamines, histones, and nonhistone proteins are pivotal to complete and maintain "maximal chromosome condensation" during mitosis.
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Materials and methods |
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Cell lines and culture
Male IM deer primary fibroblast cells (American Type Culture Collection) and the BV173 human leukemia T/B-progenitor cell line (gift from Dr. J.D. Rowley, University of Chicago, Chicago, IL) were grown in log phase from previously seeded cell cultures at 0.07 x 106 cells/ml in F10 media (Life Technologies), 10% FCS, or 0.5 x 106 cells/ml in RPMI (Life Technologies) with 10% FCS, respectively.
Cryopreservation and fracturing of cells
The cryopreservation was performed according to Chandra et al. (1986) with modifications. IM cells were grown on 99.99% pure silicon substrate discs, or fractionated mitotic cells were layered onto silicon discs and immediately dipped into a liquid N2 plus 1,1,1-trichloroethane slush bath (-150°C) at a speed of 2 m/s to a depth of 5 cm for 10 s. Cryofracturing of cells was obtained by splitting open a sandwich of two discs. The frozen discs were then transferred under N2/1,1,1-trichloroethane into a 65°C vacuum chamber for 16 h at a pressure of 0.5 millitorr and then brought to room temperature in 2 h under vacuum.
Cell synchronization
IM and BV173 cells were synchronized at the G1/S phase border using aphidicolin (20 µM) for 16 h. The block was then released for 3 h, and 0.3µM nocodazole was added for an additional 18 h. Mitotic cells were collected in the supernatant fraction, and DAPI staining showed >98% pro- or metaphase cells with only 12% interphase cells. For experiments involving chelators, we first synchronized IM cells to the G1/S border and then added 0.2 µM nocodazole for 18 h. Using this approach, >80% of the total cells were synchronized to the G2/M border, as determined by optical microscopy and DAPI staining. After releasing the G2/M block, either EGTA (5 mM) or EDTA (5 mM) or BAPTA (0.3 mM) and BAPTA-AM (50 µM) was added for 6 h, and mitotic chromosomes were harvested using the chromosome isolation methods described below for comparison.
Chromosome harvests
Two chromosome harvesting procedures were used for SIMS and IF analysis. In one method, chromosomes were extracted from mitotic cells and then fixed in methanol: acetic acid (3:1) according to Ohnuki (1968). This extraction method could lead to artifacts by contaminating chromosomes with cytosolic cations. Therefore, individual IM and BV173 metaphase chromosomes were also fractionated from cells that preserve the morphological integrity of chromosomes as shown by Saitoh and Laemmli (1994). Briefly, after cells were synchronized at mitosis, chromosomes were isolated in Ca2+- and Mg2+-free buffers using a glycerol step-gradient and then fixated with 4% p-formaldehyde. For SIMS analysis, chromosome samples were mounted onto alumina ceramic coverslips, which are devoid of innate K+, Na+, Ca2+, and Mg2+ ions, previously lightly Au coated to ensure substrate conductivity, and then air-dried. The samples were further coated with a thin sputter-deposited layer of gold to prevent electrical charging.
Immunofluorescence
IM and BV173 fractionated chromosomes were fixed with 4% p-formaldehyde and incubated with antiTopo II monoclonal (Boehringer), anti-ScII polyclonal (Saitoh et al., 1994), anti-H1 monoclonal (Biodesign), and antihCAP-C polyclonal (Schmiesing et al., 1998) antibodies, diluted 1:200, 1:50, 1:30, and 1:100, respectively, with 3% BSA in PBS. Rhodamine-conjugated antimouse or antirabbit secondary antibodies (Boehringer) were used in a dilution of 1:150. The chromosomes were analyzed with a ZEISS Axioplan microscope combined with a digital CCD camera.
Quantitation of Ca2+ and Mg2+ on chromosomes
We established a Ca2+ and Mg2+ reference for SIMS using different concentrations of Ca2+ and Mg2+ mixed with high purity agarose (Seakem Gold, FMC) as a matrix. We also established Cu2+ references for SIMS. Agarose was chosen as a carrier because of the comparable chemical structure to DNA (parallel double helix with left-handed symmetry, tightly bound water, and similar relative density to DNA, which we determined to be equal 1.869 kg/l). Drying of agarose, necessary for SIMS analysis, has only minimal impact on structure (Arndt and Stevens, 1994). The standards were solubilized in bidistilled water, deposited on Au-coated glass coverslips, vacuum dried, Au coated, and mass analyzed for Ca2+ and Mg2+ using SIMS. Control agarose samples were also SIMS analyzed, indicating negligible Ca2+ and Mg2+ background. SIMS sensitivity was greater than 10-8 M for 40Ca2+ and greater than 10-7 M for 24Mg2+ and 63Cu2+. Calibration plots of SIMS signal intensity (cts/pxl) corresponding to different cation concentrations are shown in Fig. 4, G and H. The number of metal atoms/nucleotide was obtained by multiplying the measured local metal atomic concentration (in ppm) by the average number of atoms/nucleotide (taken as 36) divided by 106.
Fluorescent filter-binding assay for the detection of Ca2+-binding proteins
The assay was performed according to Tatsumi et al. (1997), except using MgCl2 in the washing buffer. Protein marker and Calmodulin (Sigma-Aldrich), purified human Topo II (TopoGen), and fractionated IM chromosomes were electrophoresed on a 7.515% SDS gradient gel and transferred onto PVDF membrane (Bio-Rad Laboratories). After incubating the membrane with 1 mM CaCl2 and then with quin-2 (Sigma-Aldrich) for 1 h, the fluorescent proteins (Ca2+-binding proteins) were visualized by illumination with UV light at 365 nm, digitally photographed, and analyzed with the Kodak 1D Imaging system.
Topo II relaxation reaction
0.2 µg of supercoiled plasmid pSP72 (Promega) DNA was incubated at 30°C for 520 min in the presence of 1 U of purified human Topo II (TopoGen) and Ca2+ and Mg2+ in different ratios in a Topo II relaxation buffer (Osheroff and Zechiedrich, 1987). The reaction was stopped with 1% SDS and 15 mM EDTA, and the DNA phenol/chloroform was extracted, ethanol precipitated, and analyzed on a 1.5% agarose gel. The Topo II relaxation activity was quantified using the ImageQuant analysis program (Molecular Dynamics).
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Footnotes |
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* Abbreviations used in this paper: 3D, three-dimensional; AT, ademine-thymine; BAPTA, 1,2-bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid; IM, Indian muntjac; ISI, ion-induced secondary ion; PVDF, polyvinyldifluoride; SAR, scaffold-associated region; ScII, scaffold protein II; SIMS, secondary ion mass spectrometry; Topo II, topoisomerase II
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Acknowledgments |
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At the Enrico Fermi Institute (Chicago, IL), this work was supported by a grant from the Pritzker Foundation.
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References |
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