Regulation of kinetochore function by Topoisomerase II

拓扑异构酶 II 对动粒功能的调节

基本信息

批准号：
9199088
负责人：
Yoshiaki Azuma
金额：
$ 29.45万
依托单位：
UNIVERSITY OF KANSAS LAWRENCE
依托单位国家：
美国
项目类别：
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/9199088
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

项目摘要

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）：来自其衍生干细胞的精子和卵母细胞的成熟分别涉及〜400和〜30个有丝分裂分裂，而每个成人人类的生命周期约有1016个分裂。分裂期间的染色体隔离错误会导致不育，流产，出生缺陷和癌症。密切连接的机制通过固定不正确的微管 - 金属卵（MT-K）相互作用（称为误差校正），并在姐妹染色体分离之前暂停细胞周期（通过纺锤体检查点）来保护我们免受非整倍性的影响。两个过程中的主要活性是Aurora B激酶。我们已经发现了Aurora B和Topoisomerase II（Topo II）之间的新机械联系，这是一种以前仅以其链条反应而闻名的酶，从而改变了DNA拓扑。已经假定，由于其酶活性，Topo II对于染色体分离至关重要。然而，一个未解决的问题是，托波斯II在染色体的动力学上是否具有其他功能，在染色体的动物学上，托波斯II在有丝分裂和生成细胞周期检查点信号的地方最丰富。使用爪蟾鸡蛋提取物分析，我们发现TOPO II C末端结构域（CTD）的Sumoylation不会改变酶活性，而是促进染色体蛋白的特定结合。我们发现CTD Sumoylation促进了细胞周期因子的结合，这表明了TOPO II对MITOTI KINETOCHORE功能的调节的新见解。在通过Sumoypated Topo II募集到动力学的蛋白质中，有claspin和haspin调节主轴检查点激酶Aurora B.基于这一发现，我们的中心假设是“ Topo II介导的Aurora aurora b通过classpaspin和Haspin play play play play to py在有丝分裂过程中的检查点调节中的作用，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，无爪诺蛋白酶提取物细胞的测定和转基因的人类细胞系来研究这一点。我们将操纵Topo II的Sumoylation状态，以控制与Claspin和Haspin的相互作用。我们将阐明：1）TOPO II-淀粉化在通过claspin和haspin调节Aurora B活性中的作用。 2）TOPO II依赖性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，该步骤控制动力学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Kinetochore局部TOPO II作为有丝分裂检查点调节的信号分子的新功能。阐明TOPO II信号传导功能的分子机制将允许改进的TOPO II靶向疗法进行癌症治疗。