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: 10470792
• 负责人: Jocelyn Haversat
• 金额: $ 2.15万
• 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 2022-10-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470792
• 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.

有性繁殖的生物体依赖于减数分裂过程中适当的染色体分离，以确保染色体的完整性。 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 并经历染色体交换，即两个同源染色体之间的遗传信息交换。 染色体这一过程导致同系物之间形成物理连接， 染色体对在减数分裂I期间分离。交叉形成的缺陷可能是灾难性的，导致 非整倍体和诸如与年龄相关的不孕症、流产和唐氏综合症的病症。尽管 尽管这一过程非常重要，但对交叉形成的机制仍知之甚少。目标 该项目的目的是确定在减数分裂期间如何指定交换以实现 遗传信息。最近对C. elegans已经鉴定出一种细胞周期蛋白样蛋白COSA-1， 对于处理减数分裂DNA双链断裂进入交换至关重要。这一发现随后被 随后鉴定了其哺乳动物直系同源物CNTD 1，其在交叉中具有保守作用 阵然而，没有CDK被确定为COSA-1/CNTD 1的结合伴侣，以及这些细胞周期蛋白- 类似的蛋白质指定的交叉是未知的。最近，我发现C。elegans同源物 CDK 2（CDK-2）定位于交叉位点，提高了CDK 2可能与COSA-1结合的可能性。 1/CNTD 1，并作为活性激酶促进交叉形成。为了支持这一观点，哺乳动物 长期以来，CDK 2一直在交叉点以及端粒中观察到。然而，由于其端粒功能，CDK 2 基因敲除小鼠在同源配对和突触中表现出严重缺陷，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得到验证。这里我建议使用C。 elegans作为模型系统研究CDK 2在减数分裂重组中的保守功能。不像 哺乳动物CDK-2，CDK-2是同源配对和突触所必需的，但特别是对于 交叉编队在目标1中，我将使用生长素诱导的降解系统来确定 通过超分辨率显微镜对重组机器上的CDK-2耗竭。我还将决定 CDK-2和COSA-1使用纯化的组分形成活性激酶复合物。在目标2中，我将建立 CDK 2通过识别其减数分裂底物来指定交换的机制， 无偏见的化学遗传学方法。然后，我将确定CDK 2的功能意义， 通过定向诱变在C.优雅的总之，这项工作的结果将阐明保守的 指定交叉的调控机制，并将广泛适用于高等真核生物。约翰 霍普金斯大学提供了一个国家的最先进的研究环境，研究生成功的强大记录， 庞大的区域科学支持网络，以及广泛的职业发展计划，使其成为独一无二的 适合我所提出的研究和职业目标的成功。