Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: The complex multimers formed by topoisomerase IIα and HMGB1 consist of fully catenated DNA molecules. Multimers were formed with plasmid DNA of 9.1 kb (panels A–D) or 2.8 kb (panels E–F), HMGB1 and human topoisomerase IIα. The samples were deproteinized and coated with RecA protein as single-stranded (panels A–D) or double-stranded DNA (panels E–F) before visualization by electron microscopy. All negatives were shot at 50 000 magnification. The bars represent 0.1 μm.

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Plasmid Preparation, Electron Microscopy

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 stimulates decatenation of kinetoplast DNA and relaxation of supercoiled DNA by topoisomerase IIα. ( A ) Decatenation of kinetoplast DNA (kDNA) by topo IIα is stimulated by HMGB1. kDNA (0.2 μg) was incubated with increasing concentrations of topo IIα (as indicated) in the absence or presence of HMGB1 (1 μM). ( B ) Domain structure of HMGB1, HMGB1-ΔC, HMGB1 lacking the acidic C-tail (depicted as oval); A, domain A; B, domain B. ( C ) HMGB1 stimulates decatenation of kDNA by topo IIα via the HMG-box domains. kDNA (0.2 μg) was incubated with topo IIα (2 nM) in the presence or absence of increasing amounts of HMGB1, HMGB1-ΔC, domain A or domain B (as indicated). ΔC, HMGB1-ΔC. ( D ) HMGB1 stimulates relaxation of negatively supercoiled DNA by HMGB1. Relaxation of negatively supercoiled pBR322 plasmid DNA (∼8 nM) by different concentrations of topo IIα (as indicated) in the presence or absence of recombinant HMGB1 (1 μM), 37°C for 45 min. Decatenation and relaxation assays were carried out in the absence of PEG. Deproteinized DNA samples were resolved on 1% agarose gels containing ethidium bromide (A and C) or without ethidium bromide (panel D). The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. oc, nicked closed-circular DNA minicircle; rel, relaxed closed-circular DNA minicircle.

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Incubation, Plasmid Preparation, Recombinant

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: Stimulation of topo IIα activity by HMGB1 is not due to the effect of HMGB1 on DNA topology. ( A ) HMGB1 protein and structure of HMGB1 domains A and B indicating positions of the mutated intercalating amino acid residues (in red frames). ( B ) Purity of HMGB1 and mutant as revealed by electrophoresis on an SDS–18% polyacrylamide gel and Coomassie blue R-250 staining. Lane 1, wild-type HMGB1; lane 2, HMGB1 mutant (F38A/F103A/I122A). A, alanine; F, phenylalanine; I, isoleucine. ( C ) Alanine mutagenesis of intercalating residues of HMGB1 abrogated the ability of HMGB1 to introduce supercoils into DNA by topoisomerase I. Closed-circular pBR322 plasmid DNA (∼9 nM) was incubated with wheat germ topoisomerase I in the absence or presence of HMGB1 or mutant (1, 2, 3 and 6 μM, left to right). FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. (D ) Both wild-type HMGB1 and HMGB1 mutant can enhance decatenation of kDNA by human topo IIα. Decatenation of kDNA was carried out as in A. HMGB1 and mutant were at 1 μM, topo IIα was 7.5 and 15 nM. ( E ) HMGB1 does not stimulate decatenation of kDNA by E. coli topoisomerase IV. Decatenation of kDNA by E. coli topoisomerase IV (0.15 and 0.6 U) was carried out as detailed in the Materials and Methods section using HMGB1 or mutant (1 and 4 μM, left to right). The DNA samples in (C–E) were separated on 0.8% agarose gels without (C) or with (D–E) ethidium bromide in the course of electrophoresis as detailed in the Materials and Methods section. The gels are presented as negatives. WT, wild-type HMGB1; Mut, triple-mutant (F38A/F103A/I122A) of HMGB1.

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Activity Assay, Mutagenesis, Electrophoresis, Staining, Introduce, Plasmid Preparation, Incubation

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 physically interacts with topoisomerase IIα. HMGB1, HMGB1-ΔC, domains A and B (expressed in E. coli and fused proteins with GST at their N-termini) were bound to glutathione-Sepharose beads (GST was used as a negative control). The beads were then incubated with isolated human topo IIα, followed by extensive washing of the beads, and boiling them in SDS-containing buffer. The released proteins were resolved by electrophoresis on an SDS–7.5% polyacrylamide gel, followed by western blotting and immunological detection using polyclonal antibody specific to topo IIα. Input, 10% of the total topo IIα used for the pull-down assay. Upper and lower PD experiments were performed in the absence or presence of 10 U of DNAse I in the incubation buffer, respectively.

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Negative Control, Incubation, Isolation, Electrophoresis, Western Blot, Pull Down Assay

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 stimulates binding of topoisomerase IIα to DNA. 32 P-labeled 36-bp DNA duplex (∼75 nM) containing a high-affinity binding site for topo IIα was mixed with topo IIα (75 nM) and increasing amounts of HMGB1 protein (0, 0.4, 0.6, 0.8 and 1.6 μM, lanes 2–6, respectively), followed by separation of unbound DNA (free probe) and DNA–protein complexes on a 5% non-denaturing polyacrylamide gel (EMSA) as detailed in Materials and Methods section. The dried gel was subjected to PhosphorImaging. Lane 7, HMGB1 at 1.6 μM. Arrowhead indicates mobility of the topo IIα–DNA complex.

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Binding Assay, Labeling

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 and catalytic cycle of DNA topoisomerase IIα. The drawing is adapted from ref. . The ATPase domains of topo IIα (light green) and the core domains (dark green) are depicted (the C-terminal domain which is not required for the enzymatic activity of topo IIα, is not shown). Binding of two double-stranded DNA duplexes G (in red) and T (in blue) initiates the catalytic cycle (Step 1). Upon binding of two ATP molecules (red filled circles), the two ATPase domains dimerize (Step 2). Cleavage of G duplex in Step 3 (‘pre-strand passage DNA cleavage’) is followed by passage of the T duplex through the double-stranded break in the G duplex (‘Gpost-strand passage DNA cleavage’) (Step 4). Religation of the G duplex and hydrolysis of ATP proceeds in Step 5. Two molecules of ADP dissociate in Step 6, followed by release of the T duplex through the C-terminal part of topo IIα. Reopening of the ATPase domains allows dissociation of the G duplex (Step 7) to regenerate the enzyme for the next catalytic cycle. Individual steps of the topo II catalytic cycle which are stimulated the most by HMGB1 are indicated by arrows: Step 1 (DNA binding, ), Steps 3 and 4 (DNA cleavage, ). The dashed arrow indicates the HMGB1-mediated partial relief of topo IIα inhibition by ICRF-193 in Step 6 .

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Activity Assay, Binding Assay, Inhibition

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 stimulates DNA cleavage activity of topoisomerase IIα. ( A ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the absence of etoposide. DNA cleavage reactions contained negatively supercoiled plasmid pBR322 (∼4 nM), human topo IIα (∼8 nM) and increasing concentrations of HMGB1 (1, 2 and 3 μM, left to right). ATP or a non-hydrolyzable ATP analog (AMP-PNP) were at 1 mM. Arrowhead indicates position of linearized DNA. ( B ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the presence of etoposide. The molar concentrations of topo IIα and plasmid pBR322 were identical as in panel A. HMGB1 protein was at 1 μM and etoposide at 50 μM. ( C ) Effect of HMGB1 on DNA cleavage by topo IIα in the presence of increasing concentrations of etoposide. Negatively supercoiled plasmid DNA (4 nM ) was pre-incubated on ice either with buffer (control) or HMGB1 (1 μM) in the presence of 0–100 μM etoposide. Cleavage reactions were initiated by addition of topo IIα (8 nM) and incubation was carried out at 37°C for 15 min. The cleavage complexes in A–C reactions were trapped by 1% SDS, followed by digestion with proteinase K as detailed in Materials and Methods section. Deproteinized DNA samples were resolved on 1% agarose gels and subsequently stained with ethidium bromide. FIII, Hin dIII-digested plasmid pBR322 (last right lanes in A and B) indicates mobility of linearized plasmid DNA. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. The gels are presented as negatives. The percentage of cleaved plasmid DNA was quantified from three independent cleavage experiments, each in duplicates, by ImageQuant TL (GE Healthcare). The average SD for the data was <7%. DNA cleavage is expressed as percentage of initial uncleaved supercoiled plasmid pBR322. (open circle) In the absence (control) or in the presence of HMGB1 (Filled circle).

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Activity Assay, Plasmid Preparation, Incubation, Control, Staining

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 does not significantly affect DNA religation by topoisomerase IIα. DNA cleavage reactions contained negatively supercoiled plasmid DNA (4 nM ) , 50 μM etoposide in the absence (control) or presence of HMGB1 (1 μM). Cleavage reactions were initiated by addition of topo IIα (8 nM) and incubation at 37°C for 15 min. The incubation temperature was then shifted to 65°C to suppress cleavage and to promote religation as described in Materials and Methods section. A representative ethidium bromide-stained agarose gel of the DNA religation assay is shown (religation times were 0, 2.5, 5, 10, 20, 30 and 40 min, left to right). The percentage of linear DNA was estimated by the Multi Gauge software using imaging system LAS-3000 (Fuji). Each curve represents the average of 2–3 independent assays. The average SD for the data was <8%. The amount of linearized plasmid molecules in the cleavage reaction containing or lacking HMGB1 was expressed as 100%. (open circle) in the absence (control) or in the presence of HMGB1 (filled circle). FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linear plasmid DNA.

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Plasmid Preparation, Control, Incubation, Staining, Agarose Gel Electrophoresis, Software, Imaging

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in and ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO ) was measured by thin layer chromatography and quantified by PhosporImaging. Data represent the average of two independent experiments.

Article Snippet: Escherichia coli topoisomerase IV (10 U/μl) and wheat germ topoisomerase I (2–10 U/μl) were purchased from Topogen and Promega, respectively.

Techniques: Inhibition, Activity Assay, Agarose Gel Electrophoresis, Electrophoresis, Staining, Plasmid Preparation, Thin Layer Chromatography