Investigating the Role of CDK-2 in Meiotic Recombination

研究 CDK-2 在减数分裂重组中的作用

• 批准号: 10017060
• 负责人: Jocelyn Haversat
• 金额: $ 4.55万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017060
• 关键词: Address; Adult; Aneuploidy; Auxins; Binding; Biological Models; CDK2 gene; Caenorhabditis elegans; Cell Cycle Progression; Chromosome Pairing; Chromosome Segregation; Chromosomes; Complement; Complex; Coupled; Cyclin-Dependent Kinases; Cyclins; Cytology; DNA Double Strand Break; DNA Repair; Defect; Development; Down Syndrome; Ensure; Environment; Eukaryota; Exhibits; Genetic; Genetic Diseases; Genetic Recombination; Genetic Screening; Genetic Variation; Genome; Germ Cells; Goals; Homologous Gene; Human; In Vitro; Infertility; Knockout Mice; Malignant Neoplasms; Meiosis; Meiotic Prophase I; Meiotic Recombination; Microscopy; Mitosis; Mus; Mutagenesis; Nematoda; Organism; Orthologous Gene; Parents; Phosphotransferases; Process; Production; Program Development; Prophase; Proteins; Recombinant Proteins; Records; Reproduction; Research; Resolution; Role; Site; Spontaneous abortion; Sterility; System; Testing; Therapeutic Intervention; Universities; Work; age related; base; career; career development; chemical genetics; genetic approach; genetic information; in vivo; novel; offspring; success; support network; telomere; tool; transmission process

Sexually reproducing organisms rely on proper chromosome segregation during meiosis to ensure the production of gametes with the complete genetic complement. During meiotic prophase I, chromosomes pair and undergo chromosomal crossover, an exchange of genetic information between two homologous chromosomes. This process leads to the formation of physical linkages between the homologs and enables each chromosome pair to separate during meiosis I. Defects in crossover formation can be disastrous, leading to aneuploidy and conditions such as age-related infertility, miscarriages, and Down Syndrome. Despite the importance of this process, the mechanisms governing crossover formation remain poorly understood. The goal of this project is to determine how crossovers are designated during meiosis to achieve faithful transmission of genetic information. A recent genetic screen in C. elegans has identified a cyclin-like protein COSA-1 that is essential for processing meiotic DNA double-strand breaks into crossovers. This finding was subsequently followed by the identification of its mammalian ortholog CNTD1, which has conserved roles in crossover formation. However, no CDK has been identified as a binding partner of COSA-1/CNTD1, and how these cyclin- like proteins designate crossovers is not known. Recently, I have discovered that the C. elegans homolog of CDK2 (CDK-2) localizes to the sites of crossovers, raising the possibility that CDK2 might partner with COSA- 1/CNTD1 and function as an active kinase to promote crossover formation. Supporting this idea, mammalian CDK2 has long been observed at crossovers as well as telomeres. However, due to its telomeric function, CDK2 knockout mice exhibit severe defects in homolog pairing and synapsis, which are prerequisite to meiotic recombination. Therefore, the role of CDK-2 at crossover sites has remained untested. Here I propose to use C. elegans as a model system to investigate the conserved function of CDK2 in meiotic recombination. Unlike the mammalian CDK2, CDK-2 is dispensable for homolog pairing and synapsis, but is specifically required for crossover formation. In Aim 1 I will employ the auxin-inducible degradation system to determine the effect of CDK-2 depletion on recombination machineries by super-resolution microscopy. I will also determine whether CDK-2 and COSA-1 form an active kinase complex using purified components. In Aim 2 I will establish the mechanisms by which CDK2 designates crossovers by identifying its meiotic substrates through both candidate- based and unbiased chemical genetic approaches. I will then determine the functional significance of CDK2 targets through targeted mutagenesis in C. elegans. Overall, the results of this work will elucidate the conserved regulatory mechanisms that designate crossovers and will be broadly applicable to higher eukaryotes. Johns Hopkins University offers a state of-the-art research environment, strong records of post-graduate success, a vast regional scientific support network, and a wide array of career development programs that make it uniquely suited for success of my proposed research and career goals.

有性生殖生物体依靠减数分裂期间适当的染色体分离来确保 产生具有完整遗传互补的配子。在减数分裂前期 I，染色体配对 并经历染色体交叉，即两个同源基因之间遗传信息的交换 染色体。这个过程导致同系物之间形成物理连接，并使每个 染色体对在减数分裂过程中分离 I。交叉形成中的缺陷可能是灾难性的，导致 非整倍体和年龄相关不孕症、流产和唐氏综合症等疾病。尽管 虽然这一过程的重要性，但人们对交叉形成的机制仍知之甚少。目标 该项目的目的是确定减数分裂过程中如何指定交叉，以实现忠实的传递 遗传信息。最近对秀丽隐杆线虫进行的基因筛选发现了一种细胞周期蛋白样蛋白 COSA-1，它是 对于处理减数分裂 DNA 双链断裂至交叉至关重要。这一发现随后被 随后鉴定了其哺乳动物直系同源物 CNTD1，它在交叉中具有保守的作用 形成。然而，尚无 CDK 被确定为 COSA-1/CNTD1 的结合伴侣，以及这些细胞周期蛋白如何 像蛋白质指定的交叉是未知的。最近，我发现线虫的同源物 CDK2 (CDK-2) 定位于交叉位点，增加了 CDK2 可能与 COSA 合作的可能性 1/CNTD1 并作为活性激酶促进交叉形成。支持这个观点的是，哺乳动物 长期以来，人们一直在交叉和端粒中观察到 CDK2。然而，由于其端粒功能，CDK2 基因敲除小鼠在同源配对和突触方面表现出严重缺陷，这是减数分裂的先决条件 重组。因此，CDK-2 在交叉位点的作用尚未经过测试。这里我建议使用C。 线虫作为模型系统研究 CDK2 在减数分裂重组中的保守功能。与 哺乳动物 CDK2、CDK-2 对于同源配对和突触是可有可无的，但对于同源配对和突触是特别需要的 交叉形成。在目标 1 中，我将采用生长素诱导降解系统来确定 通过超分辨率显微镜观察重组机器上的 CDK-2 消耗。我也会确定是否 CDK-2 和 COSA-1 使用纯化的成分形成活性激酶复合物。在目标 2 中，我将建立 CDK2 通过识别其减数分裂底物来指定交叉的机制 基于且公正的化学遗传学方法。然后我将确定CDK2的功能意义 通过在线虫中的定向诱变来实现目标。总的来说，这项工作的结果将阐明保守的 指定交叉的调控机制将广泛适用于高等真核生物。约翰斯 霍普金斯大学提供最先进的研究环境、研究生成功的良好记录、 庞大的区域科学支持网络以及广泛的职业发展计划使其独一无二 适合我提出的研究和职业目标的成功。