Mechanisms of Trinucleotide Repeat Expansion via Oxidative DNA Damage and Repair

我获得K99/R00奖的目的是将我的研究扩展到体内DNA重复序列不稳定性、DNA损伤和修复之间的相互作用的研究，该领域是由环境应激诱导的、由碱基切除修复(BER)介导的人类疾病相关重复序列不稳定性。其长期目标是了解环境应激如何通过启动和调节重复序列不稳定性来影响人类疾病的发展和进展，以及如何通过DNA损伤修复来防止环境诱导的影响。我推测环境氧化DNA损伤及其低效误码率与CAG重复有关 扩张。这一假说将通过两个具体目标进行探索。目的一是研究环境致癌物、铬酸盐和溴酸盐对氧化单链DNA(SsDNA)断裂中间体的低效处理在CAG重复扩增中的作用。单链DNA断裂处理不足对CAG重复扩增的影响将在Pol p DRP裂解酶和发夹结构FEN1裂解缺失的情况下确定。目的二是确定氧化单链DNA断裂的高效处理是否可以防止CAG重复序列的扩张。这一目标将通过确定是否 通过APE1与POLp、XRCC1与POL3以及BLM与FEN1之间的BER蛋白相互作用，有效地处理单链DNA断裂中间产物，可以减少CAG重复序列的扩张。在威尔逊博士的指导下，在获奖期间的指导阶段，我成功地完成了我的研究和职业目标。我已经建立了几种分析CAG重复不稳定的方法 体内和体外。这提高了我在体内分析TNR稳定性和BER生化的技能和知识。这也巩固了在独立阶段实现研究目标以及构建我未来的R01提案的基础。我未来的研究将侧重于环境氧化DNA损伤引起的重复序列不稳定性的细胞和生化研究领域的工作。BER突变对三核苷酸重复序列稳定性和BER蛋白生化的影响。

My intention in obtaining ttie K99/R00 award is to extend my research into studies of in vivo interplay between DNA repeat sequence instability, DNA damage and repair in the field of human disease-associated repeat sequence instability that is induced by environmental stress and mediated by base excision repair (BER). IVIy long-term goal is to understand how exposure to environmental stress influences the development and progression of human diseases through initiating and modulating repeat sequence instability and how the environmentallv-induced effects can be prevented by DNA damage repair. I hypothesize that environmental oxidative DNA damage and its inefficient BER is involved in CAG repeat expansion. The hypothesis will be explored by two Specific Aims. Aim one is to determine how inefficient processing of oxidative single-strand DNA (ssDNA) break intermediates induced by environmental carcinogens, chromate and bromate may be involved in CAG repeat expansion. The impact of insufficient processing of ssDNA breaks on CAG repeat expansion will be determined under deficiency of Pol p dRP lyase and FEN1 cleavage of hairpin structures. Aim two is to determine if highly efficient processing of oxidative ssDNA breaks can prevent CAG repeat expansion. This aim will be examined by determining if CAG repeat expansion can be reduced by efficient processing of ssDNA break intermediates through BER protein interactions between APE1 and Pol p, XRCC1 and Pol 3, as well as BLM and FEN1. Under Dr. Wilson's mentoring, I have successfully accomplished my research and career goals during the mentored phase of the award period. I have established several approaches for analyzing CAG repeat instability in vivo and in vitro. This has advanced my skills and knowledge in analyzing in vivo TNR stability and BER biochemistry. This also consolidated the basis for fulfillment of research goals during the independent phase as well as construction of my future R01 proposals. My future research will emphasize work in the areas of cellular and biochemical studies on repeat sequence instability induced by environmental oxidative DNA damage. BER mutational effects on trinucleotide repeat stability and BER protein biochemistry.