DNA repair deficient human cells for genomic variation analysis

描述(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)的正式启动是为了系统和全面地评估基因多态如何影响我们对环境引起的疾病的易感性。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。这些类别包括细胞周期、DNA修复、细胞分裂、细胞信号、细胞结构、基因表达、凋亡和新陈代谢。基因组不断受到环境和内源性DNA损伤剂的联合攻击，需要一组复杂的DNA修复蛋白来解决这些遗传侮辱。然而，人类对DNA损伤剂的易感性存在明显的个体间差异，这些损伤剂是由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，SNPs)引起的。评估个体多态对功能的影响将需要新的方法和新的试剂。在这项建议的第一阶段，我们将开发和鉴定一系列同基因DNA糖基酶缺陷的人类细胞系，以便进一步研究DNA修复基因SNPs和遗传变异在人类细胞中的功能意义。这些研究旨在提供必要的试剂，以帮助了解人类DNA多态的生物学意义，以及这些SNPs单独或与特定环境应激因素结合在癌症、衰老相关疾病、中风和糖尿病等疾病后果中的作用。在第一阶段的成功完成后，我们打算已经证明了培育完全缺乏DNA修复蛋白，特别是DNA糖基酶的稳定的人类细胞株的可行性。此外，我们建议对每个新开发的细胞系的mRNA和蛋白质表达以及DNA糖基酶活性进行表征，最后，将评估每个新开发的细胞系单个DNA修复基因产物缺失对全球转录组的影响。为另外140个DNA修复基因开发这样的等基因人类细胞将是该提案第二阶段的主题，包括涉及碱基切除修复(BER)、直接逆转损伤、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、编辑和处理核酸酶、Rad6途径、染色质结构、疾病中存在缺陷的DNA修复基因和保守的DNA损伤反应基因。 与公共卫生相关：第一阶段项目的总体目标是开发每一种都耗尽已知DNA修复相关糖基酶的细胞系。在这项提案中，我们计划开发实时体内试验来监测糖基酶的活性。此外，我们打算确定单一糖基酶的耗尽对整体转录组的影响。

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.