Molecular mechanisms of germline DNA repair and DNA damage response

描述（由申请人提供）： 未能激活DNA损伤反应（DDR）通路允许具有未修复DNA的细胞分裂，并可能导致肿瘤的形成和增殖。受损的DNA修复可以通过形成缺失、扩增和总染色体重排来增加癌症的发病率。肿瘤进展的标志性特征是非整倍性，这是由受损的DNA损伤检查点激活和/或DNA修复引起的异常染色体分离造成的。重要的是，生殖系DNA修复和DNA损伤反应的缺陷也会导致非整倍体，并因此导致流产、出生缺陷、不育和肿瘤发生。尽管DDR和DNA修复与人类健康相关，但这些机制在分子水平上尚未完全理解。我们的目标是阐明在分子水平上维持线虫C基因组稳定性的机制。elegans是种系研究的理想模型系统，适用于分子、遗传、生物化学、细胞学和计算生物学方法。我们最近发现了HIM-20，一种未表征的D111/Gpatch结构域蛋白，也存在于人类中。HIM-20的部分缺失导致对电离辐射的敏感性和减数分裂双链断裂修复的延迟。HIM-20与交换促进蛋白共定位并相互作用。我们认为HIM-20参与DNA修复并促进交叉形成。我们将通过检查HIM-20突变体中重组的进展和输出来确定HIM-20在DNA修复中的功能机制; HIM-20突变体对DNA损伤的响应;驱动HIM-20定位模式的相互依赖性;识别其结合伴侣;并确定其体外DNA底物结合特异性。这些研究将为HIM-20及其人类直系同源物促进基因组稳定性的过程提供重要的见解。通过结合遗传学，分子，细胞学和生物化学方法，我们将确定ZTF-8的功能机制，ZTF-8是一种新的保守蛋白，我们的研究表明它与9-1-1 DDR复合物的一个组分相互作用，并且是DDR和DSB修复所必需的。这些研究将为我们理解生殖细胞中的DDR和DSB修复途径提供新的思路。最后，我们将确定线虫C染色体分离和凋亡所需的遗传相互作用网络。优雅我们将测试DNA修复和DDR基因中的基因突变的成对组合，并通过RNAi以高通量形式耗尽生殖系富集基因，该高通量形式设计并证明用于检测染色体错误分离和细胞凋亡。与每个查询相关的遗传相互作用谱的层次聚类将对基因进行排序，以识别功能相关的基因，预测生化途径和蛋白质复合物，将功能分配给未表征的基因，并揭示驱动基因组稳定性的生物网络的大规模结构。两者合计，这一应用将提供重要的新见解的分子机制调节基因组的稳定性。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Failure to activate the DNA damage response (DDR) pathway allows cells with unrepaired DNA to divide and can lead to the formation and proliferation of tumors. Impaired DNA repair can increase the incidence of cancer through the formation of deletions, amplifications, and gross chromosomal rearrangements. A hallmark feature of tumor progression is aneuploidy as a result of aberrant chromosome segregation stemming from impaired DNA damage checkpoint activation and/or DNA repair. Importantly, defects in germline DNA repair and DNA damage response also lead to aneuploidy, and as a result, to miscarriages, birth defects, infertility and tumorigenesis. Despite the relevance of both DDR and DNA repair for human health, these mechanisms are not fully understood at the molecular level. Our goal is to elucidate the mechanisms involved in maintaining genomic stability at the molecular level in the nematode C. elegans, an ideal model system for germline studies, amenable to molecular, genetic, biochemical, cytological and computational biology approaches. We have recently identified HIM-20, an uncharacterized D111/Gpatch domain containing protein also present in humans. Partial depletion of HIM-20 results in sensitivity to ionizing radiation and a delay in the repair of meiotic double strand breaks. HIM-20 colocalizes and interacts with crossover promoting proteins. We propose that HIM-20 is involved in DNA repair and acts to promote crossover formation. We will determine the mechanism of function of HIM-20 in DNA repair by examining both the progression and output of recombination in him-20 mutants; the response of him-20 mutants to DNA damage; the interdependencies driving the localization pattern of HIM-20; identifying its binding partners; and determining its DNA substrate binding specificities in vitro. These studies will provide critical insight into the procss by which HIM-20 and its human ortholog promote genome stability. Through combined genetic, molecular, cytological and biochemical approaches, we will determine the mechanism of function for ZTF-8, a novel and conserved protein that our studies have shown interacts with a component of the 9-1-1 DDR complex and is required for DDR and DSB repair. These studies will shed new light on our understanding of the DDR and DSB repair pathways in the germline. Finally, we will identify the genetic interaction network required for proper chromosome segregation and apoptosis in the nematode C. elegans. We will test pair-wise combinations of genetic mutations in DNA repair and DDR genes with depletion of germline-enriched genes by RNAi in a high-throughput format designed and proven to detect chromosome missegregation and cells undergoing apoptosis. Hierarchical clustering of genetic interaction profiles associated with each query will order genes to identify those that are functionally related, predict biochemical pathways and protein complexes, assign function to uncharacterized genes, and reveal the large-scale structure of the biological network driving genome stability. Taken together, this application will provide significant new insights into the molecular mechanisms regulating genome stability.