DNA repair deficient human cells for genomic variation analysis

描述（由申请人提供）：人类疾病是综合过程和因素的复杂相互作用的结果，包括遗传易感性和环境因素。环境基因组计划（EGP）正式启动，以系统和全面地评估遗传多态性如何影响我们对环境疾病的易感性。EGP已经确定了八类环境反应基因（ERG），这些基因已被证明对环境因子有反应。这些分类包括细胞周期、DNA修复、细胞分裂、细胞信号传导、细胞结构、基因表达、细胞凋亡和代谢。基因组受到环境和内源性DNA损伤因子的持续攻击，需要一套复杂的DNA修复蛋白来解决这些基因损伤。然而，人类对DNA损伤剂的易感性存在明显的个体间差异，这可能是由于先前存在的环境暴露或遗传因素，如序列变异或单核苷酸多态性（snp）。评估个体多态性的功能影响将需要新的方法和新的试剂。在本提案的第一阶段，我们将开发和表征一系列等基因DNA糖基酶缺陷的人类细胞系，用于未来的研究，以评估人类细胞中DNA修复基因snp和遗传变异的功能意义。这些研究旨在提供必要的试剂，以帮助理解人类DNA多态性的生物学意义，以及这些snp单独或与特定环境应激源联合在癌症、衰老相关疾病、中风和糖尿病等各种疾病结局中的作用。在第一阶段成功完成后，我们打算证明生产完全缺乏DNA修复蛋白（特别是DNA糖基化酶）的稳定人类细胞系的可行性。此外，我们建议从mRNA和蛋白质表达以及DNA糖基酶活性方面对每个新发育的细胞系进行表征，最后，每个细胞系将被评估单个DNA修复基因产物缺失对全局转录组的影响。另外140个DNA修复基因的等基因人类细胞的发展将是该提案第二阶段的主题，包括涉及碱基切除修复（BER）、损伤直接逆转、错配切除修复（MMR）、核苷酸切除修复（NER）、同源重组、非同源末端连接、核苷酸池的调节、DNA聚合酶、编辑和加工核酸酶、Rad6途径、染色质结构、疾病中有缺陷的DNA修复基因和保守的DNA损伤反应基因。

DESCRIPTION (provided by applicant): Human illness results from the complex interactions of integrated processes and factors, including genetic predispositions and environmental agents. The environmental genome project (EGP) was formally initiated to systematically and comprehensively evaluate how genetic polymorphisms impact our susceptibility to environmentally founded disease. The EGP has identified eight categories of environmentally responsive genes (ERG) that have been shown to react to environmental agents. These categories include cell cycle, DNA repair, cell division, cell signaling, cell structure, gene expression, apoptosis and metabolism. The genome is under continuous assault by a combination of both environmental and endogenous DNA damaging agents requiring a complex set of DNA repair proteins to resolve these genetic insults. However, there are clear inter-individual differences between humans in their susceptibility to DNA damaging agents that result from either pre-existing environmental exposures or genetic factors such as sequence variation or single- nucleotide polymorphisms, SNPs. Evaluating the functional impact of individual polymorphisms will require novel approaches and new reagents. During the first phase of this proposal, we will develop and characterize a series of isogenic DNA glycosylase deficient human cell lines for future studies towards evaluation of the functional significance of DNA repair gene SNPs and genetic variants in human cells. These studies are designed to provide essential reagents to aid in understanding the biological significance of human DNA polymorphisms and the role of these SNPs either alone or in combination with specific environmental stressors in disease outcomes as varied as cancer, aging-related disorders, stroke and diabetes. Upon successful completion of the 1st phase, we intend to have demonstrated the feasibility of producing stable human cell lines with complete deficiency in DNA repair proteins, specifically DNA glycosylases. Further, we propose to characterize each newly developed cell line with respect to mRNA and protein expression and DNA glycosylase activity and finally, each will be evaluated for the impact of the depletion of a single DNA repair gene product on the global transcriptome. The development of such isogenic human cells for an additional 140 DNA repair genes will be the topic of the second phase of this proposal, covering genes involved in Base Excision Repair (BER), Direct Reversal of Damage, Mismatch excision repair (MMR), Nucleotide Excision Repair (NER), Homologous Recombination, Non- homologous end-joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA repair genes defective in diseases and conserved DNA damage response genes. PUBLIC HEALTH RELEVANCE: The over all goal of the phase I project is to develop cell lines each depleted of the known DNA repair associated glycosylases. In the proposal we plan to develop real time in vivo assays to monitor glycosylase activity. Additionally we intend to determine the effect of depletion of a single glycosylases on the global transcriptome.