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：
9229056
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 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9229056
关键词：
Aneuploidy Antineoplastic Agents Apoptosis Automobile Driving Binding Biochemical Biochemical Pathway Biochemical Process Biological Biological Assay Biological Models Biological Process Caenorhabditis elegans Candidate Disease Gene Cell Nucleus Cells Characteristics Chromosomal Rearrangement Chromosome Mapping Chromosome Segregation Chromosome abnormality Chromosomes Complex Computational Biology Congenital Abnormality Crossbreeding Cytology 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 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 Impairment In Vitro Incidence Infertility Ionizing radiation Lead Light Maintenance Malignant Neoplasms Maps 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 interference screen 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 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）：项目摘要如果未能激活 DNA 损伤反应 (DDR) 途径，DNA 未修复的细胞就会分裂，并可能导致肿瘤的形成和增殖。 DNA 修复受损会通过形成缺失、扩增和染色体重排来增加癌症的发病率。肿瘤进展的一个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 修复受损而导致异常染色体分离的结果。重要的是，种系 DNA 修复和 DNA 损伤反应的缺陷也会导致非整倍体，从而导致流产、出生缺陷、不孕和肿瘤发生。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得到充分了解。我们的目标是阐明线虫线虫在分子水平上维持基因组稳定性的机制，线虫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 的部分耗尽会导致对电离辐射的敏感性以及减数分裂双链断裂修复的延迟。 HIM-20 与交叉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 损伤的反应；推动 HIM-20 本地化模式的相互依赖性；确定其约束伙伴；并在体外测定其DNA底物结合特异性。这些研究将为 HIM-20 及其人类直向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、分子、细胞学和生化方法，我们将确定 ZTF-8 的功能机制，ZTF-8 是一种新型保守蛋白，我们的研究表明它与 9-1-1 DDR 复合物的一个组成部分相互作用，并且是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线索。最后，我们将确定线虫中正确的染色体分离和细胞凋亡所需的遗传相互作用网络。我们将测试 DNA 修复和 DDR 基因中基因突变的成对组合，并通过 RNAi 以高通量形式耗尽种系富集基因，这种高通量形式旨在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排序基因来识别那些功能相关的基因，预测生化途径和蛋白质复合物，为未表征的基因分配功能，并揭示驱动基因组稳定性的生物网络的大规模结构。总而言之，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。