New roles for Topoisomerase II in meiosis

拓扑异构酶 II 在减数分裂中的新作用

• 批准号: 10626780
• 负责人: Aimee Jaramillo-Lambert
• 金额: $ 40万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626780
• 关键词: Alleles; Animal Model; Biochemical; Caenorhabditis elegans; Candidate Disease Gene; Cell division; Cells; Characteristics; Chromosome Segregation; Chromosome Structures; Chromosomes; Congenital Abnormality; DNA; Development; Diploidy; Embryo; Embryonic Development; Ensure; Enzymes; Female infertility; Fertilization; Generations; Genes; Genetic Recombination; Genetic Techniques; Genetic Transcription; Germ Cells; Goals; Haploidy; Human; Infertility; Male Infertility; Meiosis; Mitosis; Molecular; Morphology; Mutation; Mutation Analysis; Oogenesis; Phenotype; Play; Process; Proteins; Regulation; Research; Role; Sex Differences; Specificity; Spermatogenesis; Spontaneous abortion; Synaptonemal Complex; System; Time; Topoisomerase II; Work; ds-DNA; egg; in vivo; mutant; novel; programs; segregation; sex; sexual dimorphism; sperm cell; tyrosyl-DNA phosphodiesterase

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.

项目总结/摘要 单倍体配子（即卵子和精子）是由二倍体前体细胞分裂产生的。 减数分裂卵子和精子是高度特化的细胞，它们的形态和功能都不同。 对受精和胚胎发生的贡献。为了实现这些差异，产生了卵子和精子 来自具有性别特异性特征的减数分裂程序。然而，令人惊讶的是， 定义性别特异性的分子机制。此前，我们发现了C的一个新等位基因。elegans 拓扑异构酶II独特地破坏减数分裂期间同源染色体的分离 精子发生而不是卵子发生的分裂。拓扑异构酶II（Topo II）是一种酶， 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 一种大的ATP依赖性同二聚体酶。每个亚基断裂一条DNA链， 完整的线穿过断裂处，然后重新密封断裂处。因此，Topo II酶解决了拓扑 在复制、转录、染色体分离和重组过程中出现的问题。的 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这种酶的差异调节。 两个减数分裂程序。我们的长期目标是了解分子和系统，确保每个 卵子和精子在减数分裂期间接受正确数目的染色体。为了理解这个基本的 过程中，我们将利用后生动物模型C。elegans，其中，除了性二态减数分裂 程序，提供了许多实验优势，如快速生成时间，透明的身体，在 减数分裂的体内分析和单个top-2基因。本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相关的程序。方案1将 鉴定染色体轴成分、联会复合体（SC）解体 在第一次减数分裂中同源染色体分离之前染色体紧密化 师.使用靶向候选基因方法和突变分析，包括我们先前鉴定的 性别特异性的前2位等位基因，我们将确定差异调节SC解体和染色体的基因， 精子发生和卵子发生中的致密化。然后，在程序2中，我们深入研究了 调节TOP-2在精子发生和卵子发生中的定位和活性。这项工作建立在我们的发现之上 酪氨酰DNA磷酸二酯酶2（TDPT-1）内的突变可以抑制前2位突变表型。 使用生物化学和遗传学技术的结合，我们将确定抑制的机制， tdpt-1突变抑制子的top-2（it 7）胚胎致死性，并鉴定新的TOP-2相互作用蛋白 在有丝分裂与减数分裂中以及在精子发生与卵子发生中。