Genomic Stability Functions of SMC Proteins in Germ Cells

生殖细胞中 SMC 蛋白的基因组稳定性功能

• 批准号: 8499369
• 负责人: Raymond C Chan
• 金额: $ 28.51万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8499369
• 关键词: Address; Aneuploidy; Biological; Biological Assay; Caenorhabditis elegans; Candidate Disease Gene; Cells; Chromatids; Chromosomal Rearrangement; Chromosome Segregation; Chromosomes; Chromosomes, Human, Pair 5; Complement; Complex; Congenital Abnormality; Cytology; DNA; DNA Double Strand Break; DNA Repair; DNA Sequence; DNA Sequence Rearrangement; DNA lesion; Defect; Disease; Double Strand Break Repair; Event; Failure; Fertility; Frequencies; Gene Conversion; Generations; Genes; Genetic; Genetic Models; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Genomics; Germ Cells; Hereditary Disease; Homologous Gene; Human; Human Genetics; Infertility; Inherited; Light; Liquid Chromatography; Maintenance; Malignant Neoplasms; Measures; Mediating; Meiosis; Methods; Mitosis; Mitotic; Modeling; Molecular; Mutation; Mutation Spectra; Nematoda; Oligonucleotide Microarrays; Outcome; Outcome Study; Pathway interactions; Phenotype; Predisposition; Pregnancy loss; Premature aging syndrome; Process; Proteins; Regulation; Relative (related person); Risk; Role; Sister; Sister Chromatid; Structure; System; Testing; Variant; Y Chromosome; Yeasts; base; cell type; comparative genomic hybridization; developmental disease; egg; gene function; genome-wide; homologous recombination; mutant; novel; offspring; prevent; programs; protein complex; recombinational repair; repaired; segregation; sperm cell; tandem mass spectrometry

DESCRIPTION (provided by applicant): The loss of genomic stability in germ cells contributes to fertility and birth defects, and genomic disorders in human. Maintaining genomic stability requires accurate repair of DNA damage. A poorly- defined form of meiotic DNA double-strand break (DSB) repair is homologous recombination between sister chromatids. In humans, the mis-regulation of meiotic sister-chromatid recombination is thought to generate disease-associated rearrangements of the Y chromosome (Lange et al., Cell 138, 855 (2009)). A major barrier to studying this crucial repair process is the lack of experimental systems to specifically interrogate sister-chromatid recombination in meiosis, because there is frequent recombination occurring between homologous chromosomes. We are combining genetics and cytology in the roundworm Caenorhabditis elegans for the focused study of meiotic sister-chromatid recombination. We found the conserved SMC-5/6 protein complex functions specifically in meiotic sister-chromatid recombination. In the absence of SMC-5/6 function, inter-homolog recombination was unaffected, but inter-sister recombination was impaired leading to chromosome fragmentation. Due to a unique attribute of C. elegans chromosomes, the fragmentation defect did not result in mis-segregation. Consequently, we recovered viable offspring that showed gradual loss of germ cell immortality as later generations of offspring eventually became infertile. This project will utilize the C. elegans SMC-5/6 model to define the genetic pathway(s) that regulate meiotic inter-sister recombination, so that we can better explain how mis-regulation of this process might occur in humans. We identified candidate genes involved in this process based on the homologous recombination defects of the smc-5/6 mutants and the current models for homologous recombination repair. To complement this candidate gene approach, we will purify the SMC-5/6 protein complex to identify additional potentially novel candidate factors involved in inter-sister repair. These candidate genes will be tested for functions specifically in meiotic inter-sister recombination. Candidate genes will be systematically inactivated in genetic mutant backgrounds that only permit homolog-independent repair, and then specifically interrogated for homologous recombination repair using established cytological methods. For the second Aim of this project, we will directly define the types and frequency of mutations arising in the smc-5 and smc-6 mutants. We will perform a comprehensive analysis of DNA lesions accumulating at a defined genetic locus, using a powerful genetic system to identify de novo mutations in the unc-93 gene. This approach should allow us to identify any type of mutations. As a complementary approach we will perform genome-wide array Comparative Genomic Hybridization (aCGH) analysis to detect genomic duplication and deletion events. The combination of these two approaches will provide an unbiased test for whether the loss of SMC-5/6 function contributes to aberrant genomic rearrangements.

描述（由申请方提供）：生殖细胞中基因组稳定性的丧失导致生育力和出生缺陷以及人类基因组疾病。维持基因组稳定性需要精确修复DNA损伤。减数分裂DNA双链断裂（DSB）修复的一种定义不清的形式是姐妹染色单体之间的同源重组。在人类中，减数分裂姐妹染色单体重组的错误调节被认为产生Y染色体的疾病相关重排（Lange等人，Cell 138，855（2009））。研究这一关键修复过程的一个主要障碍是缺乏专门询问减数分裂中姐妹染色单体重组的实验系统，因为同源染色体之间经常发生重组。我们结合遗传学和细胞学在线虫的减数分裂姐妹染色单体重组的重点研究。我们发现保守的SMC-5/6蛋白复合体在减数分裂姐妹染色单体重组中特异性地发挥功能。在SMC-5/6功能缺失的情况下，同源物间重组不受影响，但姐妹间重组受损，导致染色体片段化。由于C. elegans染色体上的片段化缺陷不会导致误分离。因此，我们恢复了可存活的后代，这些后代表现出生殖细胞永生性的逐渐丧失，因为后代的后代最终变得不育。 该项目将利用C。elegans SMC-5/6模型来定义调节减数分裂姐妹间重组的遗传途径，以便我们可以更好地解释该过程的错误调节如何在人类中发生。我们根据smc-5/6突变体的同源重组缺陷和目前的同源重组修复模型确定了参与这一过程的候选基因。为了补充这种候选基因的方法，我们将纯化SMC-5/6蛋白复合物，以确定其他潜在的新的候选因子参与姐妹间修复。将测试这些候选基因在减数分裂姐妹间重组中的特异性功能。候选基因将在仅允许同源物非依赖性修复的遗传突变体背景中系统地失活，然后使用已建立的细胞学方法特异性地询问同源重组修复。对于本项目的第二个目标，我们将直接定义smc-5和smc-6突变体中出现的突变类型和频率。我们将使用强大的遗传系统来识别unc-93基因的从头突变，对在特定遗传位点积累的DNA损伤进行全面分析。这种方法应该允许我们识别任何类型的突变。作为一种补充方法，我们将进行全基因组阵列比较基因组杂交（aCGH）分析，以检测基因组重复和缺失事件。这两种方法的组合将为SMC-5/6功能的丧失是否有助于异常基因组重排提供无偏的测试。