Genomic Stability Functions of SMC Proteins in Germ Cells

描述(由申请人提供)：生殖细胞中基因组稳定性的丧失会导致人类的生育和出生缺陷，以及基因组疾病。维持基因组的稳定需要准确地修复DNA损伤。减数分裂DNA双链断裂(DSB)修复的一种模糊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了与疾病相关的Y染色体重排(Lange等人，细胞138,855(2009))。研究这一关键的修复过程的一个主要障碍是缺乏实验系统来专门询问减数分裂中的姐妹染色单体重组，因为同源染色体之间经常发生重组。我们正在结合遗传学和细胞学对秀丽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蛋白复合体在减数分裂姐妹染色单体重组中具有特异性功能。在SMC-5/6功能缺失的情况下，同源基因间重组不受影响，但姐妹间重组受阻，导致染色体断裂。由于线虫染色体的独特属性，这种片段化缺陷不会导致错误分离。结果，我们恢复了可以存活的后代，这些后代表现出生殖细胞永生能力的逐渐丧失，因为后代最终变得不育。本项目将利用线虫SMC-5/6模型来定义调控减数分裂姐妹间重组的遗传途径(S)，以便我们能够更好地解释这一过程如何在人类中发生错误调节。我们根据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这一过程的候选基因。为了补充这一候选基因的方法，我们将纯化SMC-5/6蛋白复合体，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专门的功能测试。候选基因将在仅允许同源无关修复的遗传突变背景中系统失活，然后使用已建立的细胞学方法专门询问同源重组修复。对于这个项目的第二个目标，我们将直接定义SMC-5和SMC-6突变体中出现的突变类型和频率。我们将使用一个强大的遗传系统来识别UNC-93基因的从头突变，从而对在一个定义的遗传位点上积累的DNA损伤进行全面的分析。这种方法应该可以让我们识别任何类型的突变。作为补充，我们将进行全基因组阵列比较基因组杂交(ACGH)分析，以检测基因组复制和缺失事件。这两种方法的结合将为SMC-5/6功能缺失是否导致基因组异常重排提供一种公正的测试。

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.