1. ATP-Dependent Positive Supercoiling of DNA by 13S Condensin: A Biochemical Implication for Chromosome Condensation 
Keiji Kimura, Tatsuya Hirano 
Cell 
Volume 90, Issue 4, Pages (August 1997)
DOI: /S (00)

2. Figure 1 ATP-Dependent Positive Supercoiling of DNA by 13S Condensin
(A) Immunoaffinity purification. Peptide-eluted fractions were resolved by 7.5% SDS-PAGE and the gel was stained with silver (lane 1, load; lane 2, flow-through; and lanes 3–9, eluates). The five subunits of 13S condensin (XCAP-C, -D2, -E, -G, and -H) coeluted in fractions 4 and 5. A pool of the two fractions was used for routine assays.
(B) One-dimensional supercoiling assay. A relaxed circular DNA was incubated with an affinity-purified fraction of 13S condensin (lanes 1, 2, 5, 6, 9, and 10) or with buffer alone (lanes 3, 4, 7, 8, 11, and 12) in the presence (lanes 1, 3, 5, 7, 9, and 11) or absence (lanes 2, 4, 6, 8, 10, and 12) of ATP. The reactions contained no topoisomerase I (lanes 1–4), E. coli topoisomerase I (lanes 5–8), or calf thymus topoisomerase I (lanes 9–12). The DNAs were purified, electrophoresed on a 0.7% agarose gel, and visualized by Southern blotting. The positions of relaxed circular DNA (r) and a ladder of supercoiled DNAs (s) are indicated.
(C) Antibody blocking. Supercoiling reactions were set up with 13S condensin (lanes 2–8) or with buffer alone (lane 1) in the presence of calf thymus topoisomerase I and ATP. Before adding a relaxed circular DNA, the mixtures were preincubated with control IgG (lane 3, 0.1 μg; lane 4, 1 μg) or anti-XCAP-E peptide antibody (lane 5, 0.1 mg; lanes 6 and 7, 1 μg), and with (lanes 7 and 8) or without (lanes 1–6) the XCAP-E peptide (0.1 μg).
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3. Figure 2 The Supercoiling Reaction Products Are Positively Supercoiled
A relaxed circular DNA was incubated with an affinity-purified fraction of 13S condensin in the presence of ATP and either E. coli topoisomerase I (A) or calf thymus topoisomerase I (C). For (B) and (D), negatively supercoiled DNA was added as an internal mobility marker to the reaction products of (A) and (C), respectively. Alternatively, the mixture of deproteinized DNAs shown in (D) was further treated with calf thymus topoisomerase I (E) or E. coli topoisomerase I (F). The DNAs were purified, fractionated by two-dimensional agarose gel electrophoresis, and visualized by Southern blotting. The positions of different forms of DNA are indicated as follows: positively supercoiled (1), relaxed circular (2), negatively supercoiled (3), and nicked circular (4), and linearized (5) DNAs.
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4. Figure 3 Requirement for ATP Hydrolysis in the Supercoiling Reaction
(A) Effects of nucleotide analogs on supercoiling. A relaxed circular DNA was incubated with an affinity-purified fraction of 13S condensin (lanes 2, 4, 6, 8, 10, and 12) or with buffer alone (lanes 1, 3, 5, 7, 9, and 11) in the presence of no topoisomerase I (upper panel), E. coli topoisomerase I (middle panel), or calf thymus topoisomerase I (bottom panel). The reactions were supplemented with no nucleotide (lanes 1 and 2), 4 mM ATP (lanes 3 and 4), 4 mM ATPγS (lanes 5 and 6), 4 mM ADP (lanes 7 and 8), 4 mM GTP (lanes 9 and 10), or 4 mM AMP-PNP (lanes 11 and 12). DNA was purified, electrophoresed in a 0.7% agarose gel, and visualized by Southern blotting. The positions of relaxed circular DNA (r) and a ladder of supercoiled DNAs (s) are indicated.
(B) ATPase assay. An affinity-purified fraction of 13S condensin (columns 3–5) or buffer alone (columns 1 and 2) was assayed for ATPase activity without DNA (columns 1 and 3), with double-stranded relaxed circular DNA (columns 2 and 4), or with single-stranded DNA (column 5). ATP and hydrolyzed ADP were separated by thin layer chromatography and quantitated. The activities are shown as picomoles of ATP hydrolyzed/min/pmol of 13S condensin.
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5. Figure 4
Stoichiometric Requirement for 13S Condensin in the Supercoiling Reaction
A closed circular DNA was incubated with increasing amounts of an affinity-purified fraction of 13S condensin and a fixed amount of E. coli topoisomerase I in the presence (lanes 3–7) or absence (lanes 10–14) of ATP. As a negative control, buffer alone (lanes 2 and 9) or buffer containing the elution peptide (lanes 1 and 8) was used in the presence (lanes 1 and 2) or absence (lanes 8 and 9) of ATP. DNA was purified and subjected to the standard supercoiling assay (upper panels). The same set of reactions was set up without topoisomerase I and loaded directly onto a 0.7% agarose gel. The 13S condensin-DNA complexes were visualized by Southern blotting (lower panels). The positions of the free substrates (arrow) and the protein–DNA complexes (asterisk) are indicated. The molar ratios of 13S condensin-to-DNA present in the reaction mixtures were ∼3.8:1 (lanes 3 and 10), ∼7.5:1 (lanes 4 and 11), ∼15:1 (lanes 5 and 12), ∼30:1 (lanes 6 and 13), or ∼60:1 (lanes 7 and 14). The population of DNA that was not subject to supercoiling in high dosage of 13S condensin (upper panel, lane 7) represents the nicked circular form (see Figure 2A, position 4).
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6. Figure 5 DNA-Binding Properties of 13S Condensin
(A) Cruciform and linear duplex probes used for the DNA-binding experiments. Duplex (1) and duplex (2) have sequences identical to the north/west (N/W) and the south/east (S/E) arms, respectively, of the cruciform DNA.
(B) Cruciform-binding (protein titration). Labeled cruciform DNA was incubated with increasing concentrations of affinity-purified 13S condensin (lanes 3, 4, 5, 6, and 7) or buffer alone with (lane 1) or without (lane 2) the elution peptide. DNA binding was analyzed by an electrophoretic mobility shift assay followed by autoradiography. The protein–DNA complexes are indicated by asterisks and the free probes are indicated by arrows. The molar ratios of protein-to-DNA present in the reaction mixtures were ∼3.8:1 (lane 3), ∼7.5:1 (lane 4), ∼15:1 (lane 5), ∼30:1 (lane 6), or ∼60:1 (lane 7).
(C) Cruciform binding (competition). The DNA-binding reactions were performed at a fixed concentration of protein-to-labeled DNA (∼60:1; lanes 2–8). Increasing concentrations of unlabeled duplex DNA (lanes 3, 4, and 5) or cruciform DNA (lanes 6, 7, and 8) were added as competitors into the binding reactions. The molar ratios of labeled-to-unlabeled DNA were 1:0 (lane 2), 1:5 (lanes 3 and 6), 1:50 (lanes 4 and 7), or 1:500 (lanes 5 and 8). Neither protein nor competitor DNA was added into lane 1.
(D) DNA length dependence (fixed concentration). A fixed concentration of linear DNA probes (1 fmol/5 μl reaction) of three different lengths (600 bp, open circle; 300 bp, circle; and 100 bp, open square) was incubated with increasing concentrations of affinity-purified 13S condensin (0–100 fmol/5 μl reaction). DNA binding was analyzed
using an electrophoretic mobility shift assay and quantitated. The ratio (%) of bound/input DNA is shown.
(E) DNA length dependence (fixed weight). A fixed weight (200 pg/5 μl reaction) of linear DNA probes was used in the same DNA binding-assay as shown in (D).
(F) DNA length dependence (competition). A fixed weight (70 pg/5 μl reaction) of 100 bp (upper left panel) or 600 bp (lower left panel) labeled DNA probe was incubated with 13S condensin (200 fmol/5 μl reaction for the 100 bp probe and 100 fmol/5 μl reaction for the 600 bp probe). Increasing concentrations of unlabeled 100 bp DNA (lanes 3, 4, 5, and 6) or 600 bp DNA (lanes 7, 8, 9, and 10) were added as competitors into the binding reactions. The ratios (w/w) of labeled-to-unlabeled DNA were 1:0 (lane 2), 1:1 (lanes 3 and 7), 1:6 (lanes 4 and 8), 1:36 (lanes 5 and 9), or 1:216 (lanes 6 and 10). Neither protein nor competitor DNA was added into lane 1. The binding efficiencies were quantitated and are shown as ratio (%) of bound/input DNA (upper right panel, 100 bp probe; lower right panel, 600 bp probe; circle, competition with 100 bp; open circle, competition with 600 bp).
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7. Figure 6
Cofractionation of 13S Condensin and the Biochemical Activities
(A) Heparin-Sepharose chromatography. An affinity-purified fraction of 13S condensin was fractionated further on a heparin-Sepharose column. Fractions were resolved by 7.5% SDS-PAGE and stained with silver (a) or analyzed by immunoblotting with antibodies against condensin subunits (b). The same fractions were assayed for ATPase activity in the presence (bar) or absence (open bar) of double-stranded DNA (c), for supercoiling activity in the presence of E. coli topoisomerase I (d), for supercoiling activity in the presence of calf thymus topoisomerase I (e), or for cruciform-binding activity (f). For the ATPase assays, percent ATP hydrolysis to ADP is shown. The fractions were load (lane 1), flow-through (lane 2), 0.15 M KCl-eluate (lane 3), 0.4 M KCl-eluate (lane 4), 0.6 M KCl-eluate (lane 5), 0.8 M KCl-eluate (lane 6), and no protein (lane 0).
(B) Sucrose gradient sedimentation. An affinity-purified fraction of 13S condensin was fractionated in a 5%–20% sucrose gradient. Fractions were resolved by 7.5% SDS-PAGE followed by silver stain (a) or assayed for ATPase activity in the presence (bar) or absence (open bar) of double-stranded DNA (b). Lanes 1–15, sucrose gradient fractions; lane 16, affinity-purified fraction; lane 17, affinity-purified fraction after concentration with a carrier protein BSA (i.e., before loading onto the gradient).
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8. Figure 7 Positive Supercoiling of DNA Induced by 13S condensin.
(A) The supercoiling assay. See the text.
(B) A superhelical tension model for mitotic chromosome condensation. See the text.
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