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

描述(申请人提供)：减数分裂形成单倍体配子形成二倍体生殖系细胞。在第一次减数分裂中，姐妹染色单体保持连接，而同源染色体分离。为了准确地分离同源基因，它们必须配对，并通过至少一个交叉连接。在减数分裂过程中，至少有12种蛋白质促进了互换。其中包括Zip3，这是一种保守的减数分裂特异蛋白，含有一个无名指，是泛素和相扑E3连接酶的标志。与E3连接酶功能一致，我已经证明无名指是Zip3功能所必需的，并且Zip3经历了自我催化的翻译后修饰。我推测，zips促进的重组和染色体蛋白的翻译后修饰促进了减数分裂的交叉。该项目的长期目标是了解Zip3如何促进减数分裂互换。其具体目的是：1.证明Zip3在体外和体内均具有泛素或相扑E3连接酶活性。将纯化小鼠和酵母Zip3蛋白，并将使用纯化的成分在体外测试它们促进相扑或泛素结合的能力。初步研究表明，酿酒酵母Zip3在体内经历了自我催化的翻译后修饰。这种修饰的性质将使用分子和遗传方法来确定。2.具体目标2：分析ZipS自动机的功能。为了防止Zip3修饰，修饰后的赖氨酸残基将通过定点突变改变为精氨酸。然后将使用遗传分析、专门的DNA物理分析和免疫荧光来监测这些突变菌株的减数分裂重组和同源配对。3.酵母Zip3的体内底物鉴定。一系列候选蛋白质将被检查是否存在依赖zips的翻译后修饰。相关性：染色体配对和重组是有性繁殖和染色体修复的基础。重组缺陷与人类不孕不育、流产和遗传病，特别是癌症有关。因此，了解重组的机制和调控有助于我们更好地了解这些疾病的病因。

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.