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（由申请人提供）：生殖细胞中基因组稳定性的丧失有助于人类的生育和先天缺陷以及基因组疾病。维持基因组稳定性需要准确修复DNA损伤。减数分裂DNA双链断裂（DSB）修复的定义不明的形式是姐妹染色单体之间同源的重组。在人类中，认为减数分裂姐妹 - 染色剂重组被认为会产生与疾病相关的Y染色体的重排（Lange等，Cell 138，855（2009））。研究这一关键修复过程的主要障碍是缺乏实验系统来特异性地询问减数分裂中姐妹 - 染色质重组，因为同源染色体之间经常发生重组。我们正在结合the虫秀丽隐杆线虫中的遗传学和细胞学，以研究减数分裂姐妹 - 染色剂重组。我们发现保守的SMC-5/6蛋白复合物在减数分裂姐妹 - 染色剂重组中的功能。在没有SMC-5/6功能的情况下，血间重组不受影响，但神间重组受到损害，导致染色体碎片化。由于秀丽隐杆线虫染色体的独特属性，片段化缺陷并未导致错误分离。因此，随着后代后代最终变得不育，我们恢复了可行的后代，显示出生殖细胞永生的逐渐丧失。 该项目将利用秀丽隐杆线虫SMC-5/6模型来定义调节减数分裂间神的重组的遗传途径，以便我们可以更好地解释人类对这一过程的错误调节。我们基于SMC-5/6突变体的同源重组缺陷和当前的同源重组修复模型确定了参与此过程的候选基因。为了补充这种候选基因方法，我们将净化SMC-5/6蛋白质复合物，以确定与姐姐间修复有关的其他潜在新型候选因素。这些候选基因将在减数分裂间的神间重组中的特殊功能进行测试。候选基因将在仅允许非同源性修复的基因突变背景中系统地灭活，然后使用既定的细胞学方法专门询问以进行同源重组修复。对于该项目的第二个目标，我们将直接定义SMC-5和SMC-6突变体中突变的类型和频率。我们将使用强大的遗传系统来鉴定UNC-93基因中的从头突变，对在定义的遗传基因座上积累的DNA病变进行全面分析。这种方法应该使我们能够识别任何类型的突变。作为一种补充方法，我们将执行全基因组阵列比较基因组杂交（ACGH）分析以检测基因组复制和缺失事件。这两种方法的组合将为SMC-5/6功能的丧失是否有助于基因组重排的损失提供无偏见的测试。