Genomic Stability Functions of SMC Proteins in Germ Cells

描述（由申请人提供）：生殖细胞基因组稳定性的丧失导致人类生育和出生缺陷以及基因组疾病。维持基因组的稳定性需要精确地修复DNA损伤。减数分裂DNA双链断裂（DSB）修复的一种不明确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控被认为会产生与疾病相关的Y染色体重排（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研究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。我们将遗传学和细胞学相结合，对秀丽隐杆线虫进行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蛋白复合物在减数分裂的姐妹染色单体重组中起特殊作用。SMC-5/6功能缺失时，同源间重组未受影响，但姊妹间重组受损，导致染色体断裂。由于秀丽隐杆线虫染色体的独特属性，断裂缺陷不会导致错误分离。因此，我们恢复了有活力的后代，这些后代显示出生殖细胞不朽性逐渐丧失，因为后代最终变得不育。本项目将利用秀丽隐杆线虫SMC-5/6模型来确定调控姐妹间减数分裂重组的遗传途径，以便我们更好地解释这一过程是如何在人类中发生的。我们根据smc-5/6突变体的同源重组缺陷和现有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方法，我们将纯化SMC-5/6蛋白复合物，以确定参与姐妹间修复的其他潜在的新候选因子。这些候选基因将在减数分裂姐妹间重组中进行功能测试。候选基因将在只允许同源非依赖性修复的基因突变背景下系统地失活，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。对于这个项目的第二个目标，我们将直接定义smc-5和smc-6突变体中产生的突变类型和频率。我们将使用一个强大的遗传系统来识别unc-93基因的新生突变，对在特定遗传位点积累的DNA病变进行全面分析。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。这两种方法的结合将为SMC-5/6功能的丧失是否导致异常的基因组重排提供公正的检验。

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.