Regulation of kinetochore function by Topoisomerase II

拓扑异构酶 II 对动粒功能的调节

• 批准号: 9492249
• 负责人: Yoshiaki Azuma
• 金额: $ 3.89万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-12 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9492249
• 关键词: Address; Adult; Alleles; Anaphase; Aneuploidy; Binding; Binding Proteins; Binding Sites; Biological Assay; C-terminal; Cancer Etiology; Cell Cycle; Cell Cycle Checkpoint; Cell Extracts; Cells; Chromatin; Chromosome Segregation; Chromosomes; Congenital Abnormality; DNA; DNA Binding; DNA Topoisomerases; Data; Development; Disease; Enzymes; Eukaryota; Event; Genome; Genome Stability; Genomic Instability; Goals; Haspin; Histone H3; Human; Human Cell Line; Infertility; Kinetochores; Knowledge; Ligase; Link; Lysine; Maintenance; Malignant Neoplasms; Mammals; Mediating; Meiosis; Microtubules; Mitosis; Mitotic; Mitotic Checkpoint; Mitotic Chromosome; Mitotic spindle; Modeling; Modification; Molecular; Molecular Conformation; Oocytes; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Post-Translational Regulation; Process; Proteins; Proteomics; Reaction; Recruitment Activity; Regulation; Research; Role; Signal Transduction; Signaling Molecule; Sister Chromatid; Site; Spontaneous abortion; Stem cells; Structure; Topoisomerase; Topoisomerase II; Xenopus; Yeasts; aurora B kinase; base; cancer therapy; egg; enzyme activity; improved; insight; loss of function; mutant; novel; public health relevance; segregation; sperm cell; targeted treatment; therapeutic development; therapeutic target; transmission process; yeast genetics

DESCRIPTION (provided by applicant): Maturation of a sperm and an oocyte from their derivative stem cells involves ~400 and ~30 mitotic divisions, respectively, while there are ~1016 divisions per adult human lifetime. Chromosome segregation errors during division cause infertility, miscarriage, birth defects and cancer. Intimately linked mechanisms protect us from aneuploidy by fixing improper Microtubule-Kinetochore (MT-K) interactions (termed Error Correction) and by pausing the cell cycle (via the Spindle Checkpoint) before sister chromatid separation in anaphase. A central activity in both processes is Aurora B kinase. We have discovered a novel mechanistic link between Aurora B and Topoisomerase II (Topo II), an enzyme previously only known for its strand passage reaction, which alters DNA topology. It has been assumed that Topo II is essential for chromosome segregation due to its enzyme activity. However, an unanswered question is whether Topo II has additional functions at the kinetochores of chromosomes, where Topo II is most abundant in mitosis and where cell cycle checkpoint signals are generated. Using Xenopus egg extract assays, we found that SUMOylation of the Topo II C-terminal domain (CTD) does not alter enzyme activity but promotes specific binding of chromosomal proteins. Our discovery that CTD SUMOylation promotes the binding of cell cycle factors has revealed novel insight into the regulation of mitoti kinetochore function by Topo II. Among the proteins recruited to kinetochores by SUMOylated Topo II, are Claspin and Haspin which regulate the spindle checkpoint kinase Aurora B. Based on this finding, our central hypothesis is that "Topo II-mediated regulation of Aurora B via Claspin and Haspin plays a critical role in checkpoint regulation during mitosis and that this mechanism is conserved in eukaryotes". We will examine this by combining yeast genetics, Xenopus egg extract cell-free assays, and genetically modified human cell lines. We will manipulate the SUMOylation status of Topo II to control interaction with Claspin and Haspin. We will elucidate: 1) The role of Topo II-SUMOylation in regulation of Aurora B activity via Claspin and Haspin. 2) The role of Topo II-dependent regulation of Aurora B in mitotic checkpoint signaling. These studies will define a novel regulatory step that controls kinetochore function as a molecular generator of mitotic checkpoint signals. The proposed research will uncover the novel function of kinetochore localized Topo II as a signaling molecule for mitotic checkpoint regulation. Elucidating the molecular mechanism of this signaling function of Topo II will allow the development of improved Topo II targeted therapeutics for cancer treatment.

描述（由申请人提供）：精子和卵母细胞从其衍生干细胞的成熟分别涉及约 400 次和约 30 次有丝分裂，而每个成年人一生中有约 1016 次分裂。分裂过程中的染色体分离错误会导致不孕、流产、出生缺陷和癌症。紧密相连的机制通过修复不正确的微管-动粒 (MT-K) 相互作用（称为纠错）并在姐妹染色单体分裂后期暂停细胞周期（通过纺锤体检查点）来保护我们免受非整倍体的影响。这两个过程的核心活性是 Aurora B 激酶。我们发现了 Aurora B 和拓扑异构酶 II (Topo II) 之间的新机制联系，拓扑异构酶 II (Topo II) 是一种以前仅因其链通道反应而闻名的酶，可改变 DNA 拓扑结构。由于其酶活性，Topo II 被认为对于染色体分离至关重要。然而，一个悬而未决的问题是，Topo II 是否在染色体动粒处具有额外的功能，在有丝分裂中，Topo II 最丰富，也是产生细胞周期检查点信号的地方。 使用非洲爪蟾卵提取物测定，我们发现 Topo II C 末端结构域 (CTD) 的 SUMO 化不会改变酶活性，但会促进染色体蛋白的特异性结合。我们发现 CTD SUMO 化促进细胞周期因子的结合，这揭示了对 Topo II 调节有丝分裂着丝粒功能的新见解。 SUMOylated Topo II 募集到动粒的蛋白质包括 Claspin 和 Haspin，它们调节纺锤体检查点激酶 Aurora B。基于这一发现，我们的中心假设是“Topo II 通过 Claspin 和 Haspin 介导的 Aurora B 调节在有丝分裂期间的检查点调节中发挥着关键作用，并且这种机制在真核生物中是保守的”。我们将通过结合酵母遗传学、非洲爪蟾卵提取物无细胞测定和转基因人类细胞系来研究这一点。我们将操纵 Topo II 的 SUMOylation 状态来控制与 Claspin 和 Haspin 的相互作用。我们将阐明：1) Topo II-SUMOylation 在通过 Claspin 和 Haspin 调节 Aurora B 活性中的作用。 2) Aurora B 的拓扑 II 依赖性调节在有丝分裂检查点信号传导中的作用。这些研究将定义一种新的调控步骤，控制动粒功能作为有丝分裂检查点信号的分子发生器。拟议的研究将揭示动粒局部拓扑 II 作为有丝分裂检查点调节信号分子的新功能。阐明 Topo II 这种信号传导功能的分子机制将有助于开发改进的 Topo II 癌症治疗靶向疗法。

项目成果

Non-Catalytic Roles of the Topoisomerase IIα C-Terminal Domain.

• DOI: 10.3390/ijms18112438
• 发表时间: 2017-11-17
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Clarke DJ;Azuma Y
• 通讯作者: Azuma Y

PICH regulates the abundance and localization of SUMOylated proteins on mitotic chromosomes.

• DOI: 10.1091/mbc.e20-03-0180
• 发表时间: 2020-11-01
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Hassebroek VA;Park H;Pandey N;Lerbakken BT;Aksenova V;Arnaoutov A;Dasso M;Azuma Y
• 通讯作者: Azuma Y

MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II.

MCPH1 功能缺失会增强由 Topo II 催化抑制剂引起的有丝分裂细胞敏感性。

• DOI: 10.3390/genes11040406
• 发表时间: 2020
• 期刊: Genes
• 影响因子: 3.5
• 作者: Arroyo,María;Sánchez,Antonio;Cañuelo,Ana;Heredia-Molina,RosalíaF;Martínez-Molina,Eduardo;Clarke,DuncanJ;Marchal,JuanAlberto
• 通讯作者: Marchal,JuanAlberto

Mechanisms behind Topoisomerase II SUMOylation in chromosome segregation.

染色体分离中拓扑异构酶 II SUMO 化背后的机制。

• DOI: 10.1080/15384101.2016.1216928
• 发表时间: 2016
• 期刊: Cell cycle (Georgetown, Tex.)
• 作者: Yoshida,MakotoM;Azuma,Yoshiaki
• 通讯作者: Azuma,Yoshiaki