Microfluidics-assisted display of stretched DNA in the study of DNA repair in viv

描述（由申请人提供）：拟议研究的目标是开发和应用一种新的定量方法来测定体内DNA修复效率，适合于诊断使用，可在现成的患者材料中使用。活细胞的基因组稳定性不断受到外部、环境基因毒素以及内部、反应性代谢物和酶功能障碍的威胁。细胞已经进化出多种修复DNA损伤的机制，人们普遍认为DNA修复能力的微小遗传或表观遗传变异可能对生物体的基因组稳定性产生深远的影响。近年来，将DNA修复基因单核苷酸多态性（snp）、表达水平或甲基化状态与癌症风险或治疗结果联系起来的研究呈指数增长。了解与疾病风险相关的基因型、表观遗传或转录组特征的功能、机制意义，将加深我们对疾病过程的认识，验证相关研究，并最终为未来的临床决策提供信息。在过去的几年里，我采用了一种高分辨率的全球DNA分析工具——微流体辅助显示拉伸DNA分子——并将其用于体内DNA复制及其对人体细胞DNA损伤的反应的定量分析。在这个提案中，我的目标是将这项技术应用于一个新的，迄今尚未实现的应用-测量体内的DNA修复。我将开发、验证和应用我们的DNA拉伸技术，以小鼠原代成纤维细胞中甲基加合物的碱基切除修复（BER）作为模型系统来研究DNA修复。使用XRCC1（一种对BER至关重要的蛋白质）中剂量或突变缺陷的细胞，我将确定我的方法是否允许测量BER效率的相对较小变化。在下一阶段，我将测试是否可以应用我的技术来测量临床相关样本中的DNA修复，如人类外周血单核细胞和人类淋巴母细胞样细胞系纯合XRCC1中与癌症风险增加相关的snp。最后，我将采取措施确定我的技术在BER以外的DNA修复系统研究中的适用性。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop and apply a new quantitative method of assaying DNA repair efficiency in vivo, suitable for diagnostic use in readily available patient material. Genomic stability of living cells is continuously threatened from without, by environmental genotoxins as well as from within, by reactive metabolites and enzymatic malfunction. Cells have evolved multiple mechanisms to repair a broad spectrum of damages to DNA, and it is generally thought that small genetic or epigenetic variations in the proficiency of DNA repair may have a profound lifetime impact on genomic stability of an organism. Recent years have seen an exponential increase in studies that connect DNA repair gene single nucleotide polymorphisms (SNPs), expression levels, or methylation status to cancer risk or therapeutic outcome. Understanding the functional, mechanistic significance of the genotypic, epigenetic, or transcriptome signatures found associated with disease risk should deepen our knowledge of the disease process, validate association studies and, ultimately, inform future clinical decisions. In the past several years I have adopted a high resolution, global DNA analysis tool - microfluidics-assisted display of stretched DNA molecules - and used it for a quantitative analysis of DNA replication in vivo and its response to DNA damage in human cells. In this proposal I aim to adapt this technology to a new, thus far unrealized application -- to measure DNA repair in vivo. I will develop, validate, and apply an adaptation of our DNA-stretching technology to study DNA repair using base excision repair (BER) of methyl adducts in mouse primary fibroblasts as a model system. Using cells with dosage or mutation defects in XRCC1, a protein critical for BER, I will determine whether my approach allows measuring relatively small variations in BER efficiency. At the next stage, I will test whether I can apply my technology to measure DNA repair in clinically relevant samples such as human peripheral blood mononuclear cells and human lymphoblastoid cell lines homozygous for the SNPs in XRCC1 that are associated with increased risk of cancer. Finally, I will take steps to determine applicability of my technology to the study of DNA repair systems other than BER.