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. 2020 Sep 22;15(9):e0239466.
doi: 10.1371/journal.pone.0239466. eCollection 2020.

DNA binding activity of the proximal C-terminal domain of rat DNA topoisomerase IIβ is involved in ICRF-193-induced closed-clamp formation

Shinji Kawano  1 , Kunpei Fujimoto  1 , Kazushi Yasuda  1 , Shogo Ikeda  1
Affiliations

DNA binding activity of the proximal C-terminal domain of rat DNA topoisomerase IIβ is involved in ICRF-193-induced closed-clamp formation

Shinji Kawano et al. PLoS One. .

Abstract

DNA topoisomerase II (topo II) is an essential enzyme that regulates DNA topology by DNA cleavage and re-ligation. In vertebrates, there are two isozymes, α and β. The C-terminal domain (CTD) of the isozymes, which shows a low degree of sequence homology between α and β, is involved in each isozyme-specific intracellular behavior. The CTD of topo IIβ is supposedly involved in topo II regulation. Topo IIβ is maintained in an inactive state in the nucleoli by the binding of RNA to the 50-residue region termed C-terminal regulatory domain (CRD) present in the CTD. Although in vitro biochemical analysis indicates that the CTD of topo IIβ has DNA binding activity, it is unclear whether CTD influences catalytic reaction in the nucleoplasm. Here, we show that the proximal CTD (hereafter referred to as pCTD) of rat topo IIβ, including the CRD, is involved in the catalytic reaction in the nucleoplasm. We identified the pCTD as a domain with DNA binding activity by in vitro catenation assay and electrophoretic mobility shift assay. Fluorescence recovery after photo-bleaching (FRAP) analysis of pCTD-lacking mutant (ΔpCTD) showed higher mobility in nucleoplasm than that of the wild-type enzyme, indicating that the pCTD also affected the nuclear dynamics of topo IIβ. ICRF-193, one of the topo II catalytic inhibitors, induces the formation of closed-clamp intermediates of topo II. Treatment of ΔpCTD with ICRF-193 significantly decreased the efficiency of closed-clamp formation. Altogether, our data indicate that the binding of topo IIβ to DNA through the pCTD is required for the catalytic reaction in the nucleoplasm.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The CTD of rat topo IIβ is required for efficient in vitro catenation.
(A) Structures of rat topo IIβ and the CTD truncation mutant. (B) The relaxation assay was performed with 2-fold serially-diluted enzyme (20 = 100 fmol) and 50 ng pUC18. (C) The decatenation assay was performed with 2-fold serially-diluted enzyme (20 = 100 fmol) and 100 ng kDNA. (D) Purified FLAG-tagged proteins (100 fmol) were used for catenation assay in the presence of histone H1.0 (1, 2 and 4 μg/mL) as a DNA aggregation factor. Deproteinized samples were analyzed by 1% agarose gel. (E) Catenation assay in the presence of PEG (1, 5 and 10%). I: supercoiled DNA. II: nicked circular DNA. Brackets indicate catenanes.
Fig 2
Fig 2. The DNA binding activity of the rat topo IIβ 1201–1320 region (pCTD) contributes to efficient in vitro catenation.
(A) Scheme of rat topo IIβ CTD (1201–1614) and its truncation mutants used in this study. (B) GST-tagged proteins (100, 200, 300, 400, 500, and 600 fmol) were mixed with 5 ng supercoiled and linearized pUC18 and incubated for 30 min. The reaction mixture was subjected to 1% agarose gel containing MgCl2. (C) The relaxation assay was performed as described in Fig 1. (D) The decatenation assay was performed as described in Fig 1. Arrows indicated decatenated DNA bands. (E) The catenation assay was performed in the presence of H1.0 (1, 2, and 4 μg/mL) as described in Fig 1. (F) Catenation assay in the presence of PEG (1%, 5%, and 10%) as described in Fig 1. I: supercoiled DNA. II: nicked circular DNA. III: linearized DNA. Brackets indicate catenanes.
Fig 3
Fig 3. The pCTD of rat topo IIβ supports pre-strand passage DNA cleavage activity.
(A) DNA cleavage assay was performed in the absence of ATP. FLAG-tagged protein (50, 100, and 200 fmol) was mixed with 5 ng pUC18 in the presence of 200 μM etoposide and incubated at 37°C for 15 min. Deproteinized samples were analyzed by 1% agarose gel. (B) DNA cleavage assays were performed in the presence of AMP-PNP as described in (A). I: supercoiled DNA. II: nicked circular DNA. III: linearized DNA.
Fig 4
Fig 4. The pCTD of rat topo IIβ maintains the relaxation activity of I865A mutants.
(A) Amino acid sequence alignment of E. coli topo IV ParC subunit (164–180), S. cerevisiae topo II (825–841), H. sapiens topo IIβ (869–885) and R. norvegicus topo IIβ (857–873). Box represents the conserved isoleucine (I). (B) Relaxation assays were performed using 50 ng pUC18 and purified FLAG-tagged I865A mutants (100, 50, and 25 fmol). The DNA bands were detected by GelRed staining. (C) Relaxation assays were performed using 5 ng pUC18 and purified FLAG-tagged I865A mutants (50, 100, and 200 fmol). The DNA bands were detected by SYBR Green I. I: supercoiled DNA. II: nicked circular DNA.
Fig 5
Fig 5. FRAP analysis of FLAG-topo IIβ-EGFP.
(A) Fluorescence recovery kinetics of WT (n = 9) and ΔpCTD (n = 12) in HEK293 cells. The photobleached region is indicated by a circle. Scale bars = 5 μm. Dotted lines denote nucleoli. (B) Fluorescence recovery kinetics of WTY814S (n = 5) and ΔpCTRY814S (n = 6) in HEK293 cells. Dotted lines denote nucleoli. (C, D) Normalized fluorescence intensities are plotted versus time (s). Values represent mean ± S.D.
Fig 6
Fig 6. Formation of salt-stable closed-clamp intermediates induced by ICRF-193 treatment.
(A) HEK293 cells in a 35-mm glass-bottom dish were transfected with the FLAG-topo IIβ-EGFP expression vector. The live cells were imaged using a 40× objective lens. ICRF-193 (7 μM) was added for 15 min. Scale bar = 5 μm. (B) The clamping assay was performed as described in materials and methods. FLAG and EGFP-tagged proteins were detected by using the anti-FLAG tag antibody. (C) Relative band intensities in (B) were calculated from band densitometry using Image J software. The experiments were performed in triplicate, and the results are indicated as mean ± S.D. Circles represent individual data points. Asterisks (P < 0.001) indicate a significant deviation from WT (Student’s t-test).
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