Characterization of Zip3: a novel meiosis-specific RING E3 ligase

Zip3 的表征：一种新型减数分裂特异性 RING E3 连接酶

• 批准号: 7422309
• 负责人: Eric M Mortensen
• 金额: $ 4.69万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2009-07-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7422309
• 关键词: Arginine; Biological Assay; Chromosome Pairing; Chromosomes; DNA; Defect; Diploidy; Etiology; Fingers; Genetic Crossing Over; Genetic Recombination; Germ Cells; Goals; Haploidy; Hereditary Disease; Homologous Gene; Human; Immunofluorescence Immunologic; In Vitro; Infertility; Link; Lysine; Malignant Neoplasms; Meiosis; Meiotic Recombination; Modification; Molecular Genetics; Monitor; Mus; Nature; Post-Translational Protein Processing; Proteins; Regulation; Reproduction; Saccharomyces cerevisiae; Series; Sister Chromatid; Site-Directed Mutagenesis; Spontaneous abortion; Synaptonemal Complex; Testing; Ubiquitin; Ubiquitination; Yeasts; genetic analysis; in vivo; mutant; novel; prevent; repaired; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Meiosis forms haploid gametes form diploid germ-line cells. During the first meiotic division, sister- chromatids remain connected while homologous chromosomes separate. For homologs to be accurately segregated, they must pair and become connected by at least one crossover. At least 12 proteins promote crossing-over during meiosis. Amongst these is Zip3, a conserved meiosis-specific protein that contains a RING-finger, which is a signature of ubiquitin and SUMO E3 ligases. Consistent with E3 ligase function, I have shown that the RING-finger is essential for Zip3 function and that Zip3 undergoes autocatalytic post- translational modification. I hypothesize that ZipS-promoted post-translational modification of recombination and chromosomal proteins promotes meiotic crossing-over. The long-term goal of this project is to understand how Zip3 promotes meiotic crossing-over. The Specific Aims are: 1. To demonstrate that Zip3 has ubiquitin or SUMO E3 ligase activity in vitro an in vivo. Mouse and yeast Zip3 proteins will be purified and their ability to promote SUMO or ubiquitin conjugation will be tested in vitro using purified components. Preliminary studies indicate that S. cerevisiae Zip3 undergoes self-catalyzed post-translational modification in vivo. The nature of this modification will be determined using molecular and genetic approaches. 2. Specific Aim 2: To analyze the function ofZipS automodification. To prevent Zip3 modification, modified lysine residues will be changed to arginine using site-directed mutagenesis. Meiotic recombination and homolog pairing will then be monitored in these mutant strains using genetic analysis, specialized DNA physical assays and immunofluorescence. 3. To identify in vivo substrates of yeast Zip3. A series of candidate proteins will be examined for the presence of ZipS-dependent post-translational modification. Relevance: Chromosome pairing and recombination are fundamental to sexual reproduction and chromosome repair. Defects in recombination have been linked to human infertility, miscarriage and genetic diseases, particularly cancer. An understanding of the mechanism and regulation of recombination will therefore help us better understand the etiology of these diseases.

描述（由申请人提供）：减数分裂形成单倍体配子，形成二倍体种系细胞。在第一次减数分裂期间，姐妹染色单体保持连接，而同源染色体分离。为了准确分离同系物，它们必须通过至少一次交叉配对并连接。至少有 12 种蛋白质促进减数分裂过程中的交换。其中包括 Zip3，一种保守的减数分裂特异性蛋白，含有环指，是泛素和 SUMO E3 连接酶的特征。与E3连接酶功能一致，我已经证明环指对于Zip3功能是必需的，并且Zip3经历自催化翻译后修饰。我假设 ZipS 促进的重组和染色体蛋白的翻译后修饰促进减数分裂交换。该项目的长期目标是了解 Zip3 如何促进减数分裂交叉。具体目标是： 1. 证明 Zip3 在体外和体内具有泛素或 SUMO E3 连接酶活性。小鼠和酵母 Zip3 蛋白将被纯化，并使用纯化的成分在体外测试它们促进 SUMO 或泛素缀合的能力。初步研究表明酿酒酵母Zip3在体内发生自催化翻译后修饰。这种修饰的性质将通过分子和遗传方法来确定。 2、具体目标2：分析ZipS自动修改功能。为了防止 Zip3 修饰，修饰的赖氨酸残基将使用定点诱变更改为精氨酸。然后，将使用遗传分析、专门的 DNA 物理测定和免疫荧光来监测这些突变株的减数分裂重组和同源配对。 3. 鉴定酵母Zip3的体内底物。将检查一系列候选蛋白是否存在 ZipS 依赖性翻译后修饰。相关性：染色体配对和重组是有性生殖和染色体修复的基础。重组缺陷与人类不孕、流产和遗传疾病，特别是癌症有关。因此，了解重组的机制和调控将有助于我们更好地了解这些疾病的病因。