Molecular mechanisms of germline DNA repair and DNA damage response

种系DNA修复和DNA损伤反应的分子机制

基本信息

批准号：
8892208
负责人：
Monica P Colaiacovo
金额：
$ 32.21万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-15 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8892208
关键词：
Aneuploidy Antineoplastic Agents Apoptosis Automobile Driving Binding Biochemical Biochemical Pathway Biochemical Process Biological Biological Assay Biological Models Biological Process Caenorhabditis elegans Cell Nucleus Cells Characteristics Chromosomal Rearrangement Chromosome Mapping Chromosome Segregation Chromosome abnormality Chromosomes Complex Computational Biology Congenital Abnormality DNA DNA Binding DNA Binding Domain DNA Damage DNA Double Strand Break DNA Repair DNA Repair Gene DNA Repair Pathway DNA Sequence Alteration DNA damage checkpoint DNA repair protein Defect Devices Double Strand Break Repair Dyes Excision Failure Flow Cytometry Frequencies Gene Order Genes Genetic Genetic Nondisjunction Genetic Recombination Genome Stability Genomic Instability Goals Health Homologous Gene Human Hybrids In Vitro Incidence Infertility Ionizing radiation Lead Light Maintenance Malignant Neoplasms Mediating Meiosis Mitotic Molecular Molecular Genetics Monitor Nematoda Organism Orthologous Gene Output Pathway interactions Pattern Pharmacotherapy Phenotype Play Predisposition Process Prophase Proteins RNA Interference RNA-Binding Proteins Regulation Reporter Reproductive Health Role Signal Transduction Specificity Spontaneous abortion Structure System Testing Tumor Suppressor Proteins Two-Hybrid System Techniques Yeasts Zinc Fingers base cancer diagnosis cell injury design gene discovery genome integrity high throughput screening in vivo insight member mutant novel protein complex repaired response stem tumor tumor DNA tumor progression tumorigenesis

项目摘要

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.

描述（由申请人提供）：激活DNA损伤响应（DDR）途径的项目摘要失败使具有未修复的DNA的细胞可以分裂，并可能导致肿瘤的形成和增殖。 DNA修复受损可以通过形成缺失，扩增和总体染色体重排来增加癌症的发生率。肿瘤进展的标志性特征是非整倍性，这是由于DNA损伤检查点激活和/或DNA修复受损的异常染色体隔离而导致的。重要的是，种系DNA修复和DNA损伤反应的缺陷也导致非整倍性，因此流产，出生缺陷，不育和肿瘤发生。尽管DDR和DNA修复对人类健康的相关性，但在分子水平上尚未完全了解这些机制。我们的目标是阐明在线虫C.秀丽隐杆线虫中维持基因组稳定性的机制，线虫秀丽隐杆线虫是一种适合分子，遗传，生化，细胞学和计算生物学方法的理想模型系统。我们最近确定了他20，这是一个未表征的D111/GPATCH结构域，其中包含蛋白质也存在于人类中。 HIM-20的部分耗竭会导致对电离辐射的敏感性和减数分裂双链断裂的修复延迟。 HIM-20共定位并与促进蛋白质的交叉相互作用。我们建议HIM-20参与DNA修复，并作为促进跨界形成的行为。我们将通过检查HIM-20突变体重组的进展和输出来确定HIM-20在DNA修复中的功能机理；他20突变体对DNA损伤的反应；相互依存的驱动了他20的本地化模式；确定其约束伙伴；并在体外确定其DNA底物结合特异性。这些研究将对HIM-20及其人类直系同源的Procss进行批判性见解。通过结合的遗传，分子，细胞学和生化方法，我们将确定ZTF-8的功能机理，这是一种新型和保守的蛋白质，我们的研究表明，我们的研究表明，与9-1-1 DDR复合物的成分相互作用，并且需要DDR和DSB修复。这些研究将为我们对种系中DDR和DSB修复途径的理解提供新的启示。最后，我们将确定线虫秀丽隐杆线虫中适当的染色体分离和凋亡所需的遗传相互作用网络。我们将在DNA修复中的基因突变和DDR基因中的基因组合对RNAi耗尽富含生殖线的基因，以高通量格式设计和经过证明，可检测旨在检测染色体错误隔离和细胞的细胞。与每个查询相关的遗传相互作用谱的分层聚类将订购基因，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，并揭示生物网络驱动基因组稳定性的大规模结构。综上所述，该应用将为调节基因组稳定性的分子机制提供重要的新见解。