Regulation of kinetochore function by Topoisomerase II

描述(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，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。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)和在姐妹染色单体后期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体的伤害。在这两个过程中的一个中心活动是Aurora B激酶。我们发现了Aurora B和拓扑异构酶II(Topo II)之间的一种新的机制联系，Topo II是一种以前只以链传递反应而闻名的酶，它可以改变DNA的拓扑结构。由于Topo II的酶活性，人们一直认为Topo II是染色体分离所必需的。然而，一个悬而未决的问题是，Topo II是否在染色体着丝点具有额外的功能，在那里Topo II是有丝分裂最丰富的地方，也是细胞周期检查点信号产生的地方。利用非洲爪蛙卵提取液检测，我们发现TOPO II C-末端结构域(CTD)的SUMO化不会改变酶的活性，但会促进染色体蛋白的特异性结合。我们的发现CTD SUMO化促进了细胞周期因子的结合，揭示了Topo II对有丝分裂动粒功能调控的新见解。在SuMOylated Topo II招募到动粒的蛋白质中，有Claspin和Haspin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在有丝分裂过程中的检查点调节中起关键作用，并且这一机制在真核生物中是保守的。我们将结合酵母遗传学、非洲爪哇卵提取液无细胞分析和转基因人类细胞株来检验这一点。我们将操纵Topo II的SUMO化状态来控制与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)Topo II依赖的Aurora B调控在有丝分裂检查点信号中的作用。这些研究将定义一个新的调控步骤，控制着动粒作为有丝分裂检查点信号的分子生成器的功能。这项拟议的研究将揭示动粒定位的Topo II作为有丝分裂检查点调控的信号分子的新功能。阐明Topo II这种信号功能的分子机制将有助于开发改进的Topo II靶向治疗癌症的药物。

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.