Regulation of kinetochore function by Topoisomerase II

描述（由申请人提供）：精子和卵母细胞从其衍生干细胞中成熟分别涉及~400次和~30次有丝分裂，而每个成年人一生中约有1016次分裂。分裂过程中染色体分离错误会导致不育、流产、出生缺陷和癌症。紧密联系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）和在后期姐妹染色单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。极光B激酶是这两个过程的中心活性。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，拓扑异构酶II以前只知道它的链通过反应，改变DNA拓扑结构。据推测，拓扑异构酶II是必不可少的染色体分离，由于其酶的活性。然而，一个未回答的问题是，Topo II是否在染色体的动粒上具有额外的功能，其中Topo II在有丝分裂中最丰富，并且在细胞周期检查点信号产生的地方。 使用非洲爪蟾卵提取物测定，我们发现，SUMO化的拓扑II C-末端结构域（CTD）不改变酶的活性，但促进染色体蛋白的特异性结合。我们的发现，CTD SUMO化促进细胞周期因子的结合，揭示了新的见解线粒体动粒功能的调节拓扑异构酶II。在SUMO化Topo II募集到着丝粒的蛋白质中，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“通过Claspin和Haspin的拓扑II介导的极光B调节在有丝分裂期间的检查点调节中起关键作用，并且该机制在真核生物中是保守的”。我们将结合酵母遗传学，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SUMO化状态以控制与Claspin和Haspin的相互作用。我们将阐明：1）Topo II-SUMO化在通过Claspin和Haspin调节Aurora B活性中的作用。2)Aurora B在有丝分裂检查点信号传导中的Topo II依赖性调节作用这些研究将定义一个新的调控步骤，控制动粒功能作为有丝分裂检查点信号的分子发生器。这项研究将揭示动粒定位的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的这种信号传导功能的分子机制将允许开发用于癌症治疗的改进的Topo II靶向治疗剂。

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.