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

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, ? and ?. The C-terminal domain (CTD) of the isozymes, which shows a low degree of sequence homology between ? and ?, is involved in each isozyme-specific intracellular behavior. The CTD of topo II? is supposedly involved in topo II regulation. Topo II? 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 II? 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 II?, 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 (?pCTD) showed higher mobility in nucleoplasm than that of the wild-type enzyme, indicating that the pCTD also affected the nuclear dynamics of topo II?. ICRF-193, one of the topo II catalytic inhibitors, induces the formation of closed-clamp intermediates of topo II. Treatment of ?pCTD with ICRF-193 significantly decreased the efficiency of closed-clamp formation. Altogether, our data indicate that the binding of topo II? to DNA through the pCTD is required for the catalytic reaction in the nucleoplasm.

SUBMITTER: Kawano S

PROVIDER: S-EPMC7508362 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Kawano Shinji S Fujimoto Kunpei K Yasuda Kazushi K Ikeda Shogo S

PloS one 20200922 9

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 r ...[more]

PMID: 32960919

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Project description:BackgroundXp11.2 translocation renal cell carcinoma (tRCC) is defined by translocation of the transcription factor E3 (TFE3) gene located on chromosome Xp11.2. Due to the high incidence in women, 17β-estradiol (E2) may be a factor influencing TFE3 breaks, and the topoisomerase II (TOP2) poison is considered one of the important risk factors in mediating DNA breaks. In this study, we investigated the potential pathogenesis of Xp11.2 tRCC using the renal epithelial cell line HK-2.MethodsImmunofluorescence assay was performed to analyze DNA breaks by quantifying phosphorylation of H2AX (γH2AX), and the micronuclei (MN) assay was designed for monitoring chromosome breaks. The chromatin immunoprecipitation (CHIP) was used to detect whether proteins bound to specific DNA site, and the co-immunoprecipitation (Co-IP) was used to confirm whether proteins bound to other proteins. In some experiments, siRNA and shRNA were transfected to knockdown target genes.ResultsOur results demonstrated that DNA double-strand breaks were mediated by TOP2β in HK-2 cells, and this process could be amplified through estrogen receptor α (ERα)-dependent pathway induced by E2. After performing translocation site analysis using target region sequencing data in two Xp11.2 tRCC cell lines and ten Xp11.2 tRCC patients, we confirmed that TOP2β and ERα could both bind to TFE3 translocation sites directly to mediate DNA breaks in a E2-dependent manner. However, TOP2β and ERα were not observed to have direct interaction, indicating that their collaborative may be implemented in other ways. Besides, TFE3 was found to be upregulated through NRF1 with increasing E2 concentration, which could increase the risk of TFE3 breaks.ConclusionOur results indicate that E2 amplifies TOP2β-mediated TFE3 breaks by ERα-dependent pathway, and E2 upregulates TFE3 by NRF1 to increase the risk of TFE3 breaks. This suggests that E2 is an important pathogenic factor for Xp11.2 tRCC pathogenesis. Video Abstract.

Dataset Information

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

Publications

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

Similar Datasets

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