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成熟分别涉及~400和~30个有丝分裂，而每个成年人一生中有~1016个分裂。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。密切相关的机制通过修复不适当的微管-着丝粒（MT-K）相互作用（称为纠错）和在后期姐妹染色单体分离前暂停细胞周期（通过纺锤体检查点）来保护我们免受非整倍体的侵害。这两个过程的中心活动是极光B激酶。我们已经发现了极光B和拓扑异构酶II （Topo II）之间的一种新的机制联系，这种酶以前只知道它的链传递反应，它改变了DNA的拓扑结构。由于Topo II的酶活性，它一直被认为是染色体分离所必需的。然而，一个悬而未决的问题是Topo II是否在染色体的着丝点上具有额外的功能，在有丝分裂中Topo II最丰富，并且在细胞周期检查点信号产生的地方。通过对爪蟾卵提取物的分析，我们发现Topo II c末端结构域（CTD）的SUMOylation不会改变酶的活性，但会促进染色体蛋白的特异性结合。我们发现CTD SUMOylation促进了细胞周期因子的结合，揭示了Topo II对有丝分裂着丝点功能的调控。在summoylated Topo II募集到着丝点的蛋白质中，有调节纺锤体检查点激酶Aurora B的Claspin和Haspin。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在有丝分裂过程中的检查点调节中起着关键作用，并且这一机制在真核生物中是保守的”。我们将通过结合酵母遗传学、爪蟾卵提取物无细胞测定和转基因人类细胞系来检验这一点。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调控Aurora B活性中的作用。2)Topo ii依赖性调控Aurora B在有丝分裂检查点信号传导中的作用。这些研究将定义一个新的调控步骤，控制着丝点作为有丝分裂检查点信号的分子发生器的功能。本研究将揭示着丝点定位Topo II作为有丝分裂检查点调控信号分子的新功能。阐明Topo II的这种信号功能的分子机制将允许开发改进的Topo II靶向治疗癌症。

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