New roles for Topoisomerase II in meiosis

项目摘要/摘要 单倍体配子(即卵子和精子)是由二倍体前体通过细胞分裂产生的 减数分裂。卵子和精子是高度特化的细胞，它们的形态和精子的 对受精和胚胎发育的贡献。为了实现这些差异，产生了卵子和精子 来自具有特定性别特征的减数分裂程序。然而，令人惊讶的是，人们对 定义性别专一性的分子机制。此前，我们发现了一种新的线虫等位基因 在减数分裂过程中唯一破坏同源染色体分离的拓扑异构酶II 精子发生的分裂而不是卵子发生的分裂。拓扑异构酶II(Topo II)是一种在生物体内起关键作用的酶。 通过解开双链DNA中出现的拓扑问题，在染色体保真度中发挥作用。Topo II是 一种依赖于三磷酸腺苷的大型同源二聚体酶。每个亚基断裂一条DNA链，传递第二条DNA链 未断线穿过断口，然后重新密封断口。因此，Topo II酶解决了拓扑问题 在复制、转录、染色体分离和重组过程中出现的问题。这个 对这种关键的、普遍存在的酶的性别特异性作用的鉴定突出了对 两个减数分裂程序。我们的长期目标是了解分子和系统，以确保每个 卵子和精子在减数分裂过程中获得正确数目的染色体。要理解这一基本原理 过程中，我们将利用后生动物模型线虫，它除了有性二态减数分裂外 程序，提供了许多实验优势，如快速生成时间，透明主体，用于在 减数分裂的活体分析，以及一个单一的top-2基因。这项提案中的研究包括两个主要方面 与减数分裂染色体结构和分离的性别特异性调节有关的方案。计划1将 确定在染色体轴成分、联会复合体(SC)分解、 和在第一次减数分裂中同源染色体分离之前的染色体紧致 组织。使用有针对性的候选基因方法和突变分析，包括我们之前确定的 性别特异的Top-2等位基因，我们将识别差异调控SC拆解和染色体的基因 精子发生和卵子发生中的致密化。然后，在节目2中，我们深入探讨了 在精子发生和卵子发生中调节top-2的定位和活性。这项工作是以我们的发现为基础的 酪氨酰DNA磷酸二酯酶2(TDPT-1)内的突变可以抑制TOP-2突变表型。 利用生化和基因技术的结合，我们将确定抑制 TOP-2(It7)对tdpt-1突变抑制子的胚胎致死性及鉴定新的top-2相互作用蛋白 在有丝分裂与减数分裂以及精子发生与卵子发生之间。

PROJECT SUMMARY/ABSTRACT Haploid gametes (i.e. eggs and sperm) are generated from diploid precursors through the cell division of meiosis. Eggs and sperm are highly specialized cells that differ in both their morphologies and in their contributions to fertilization and embryogenesis. To achieve these differences, eggs and sperm are generated from meiotic programs with sex-specific characteristics. However, surprisingly little is known about the molecular mechanisms that define sex-specificity. Previously, we identified a novel allele of C. elegans topoisomerase II that uniquely disrupts the segregation of homologous chromosomes during the meiotic divisions of spermatogenesis but not oogenesis. Topoisomerase II (Topo II) is an enzyme that plays a crucial role in chromosome fidelity by disentangling topological problems that arise in double stranded DNA. Topo II is a large ATP-dependent, homodimeric enzyme. Each subunit breaks one DNA strand, passes a second unbroken strand through the break, and then reseals the break. Thus, Topo II enzymes solve topological problems that arise during replication, transcription, chromosome segregation, and recombination. The identification of a sex-specific role for this key, ubiquitous enzyme highlights the differential regulation of the two meiotic programs. Our long-term goal is to understand the molecules and systems that ensure that each egg and sperm receive the correct number of chromosomes during meiosis. To understand this fundamental process, we will utilize the metazoan animal model C. elegans, which, in addition to sexually dimorphic meiotic programs, provides many experimental advantages such as a fast generation time, a transparent body for in vivo analysis of meiosis, and a single top-2 gene. The research in this proposal encompasses two main programs related to sex-specific regulation of meiotic chromosome structure and segregation. Program 1 will identify sex-specific differences in chromosomal axes components, synaptonemal complex (SC) disassembly, and chromosome compaction prior to the segregation of homologous chromosomes during the first meiotic division. Using a targeted candidate gene approach and mutational analysis, including our previously identified sex-specific top-2 allele, we will identify genes that differentially regulate SC disassembly and chromosome compaction in spermatogenesis and oogenesis. Then, in Program 2, we delve into the mechanisms that regulate TOP-2 localization and activity in spermatogenesis and oogenesis. This work builds on our findings that mutations within tyrosyl DNA phosphodiesterase 2 (TDPT-1) can suppress the top-2 mutant phenotypes. Using a combination of biochemical and genetic techniques we will identify the mechanism of suppression of top-2(it7) embryonic lethality for the tdpt-1 mutant suppressors and identify novel TOP-2 interacting proteins during mitosis vs. meiosis and in spermatogenesis vs. oogenesis.