Mechanisms of Trinucleotide Repeat Expansion via Oxidative DNA Damage and Repair

我获得K99/R 00奖的目的是将我的研究扩展到DNA重复序列不稳定性、DNA损伤和修复之间的体内相互作用，在人类疾病相关的重复序列不稳定性领域，该不稳定性由环境应激诱导并由碱基切除修复（BER）介导。长期目标是了解暴露于环境压力如何通过启动和调节重复序列不稳定性影响人类疾病的发展和进展，以及如何通过DNA损伤修复来预防环境诱导的影响。我假设环境氧化DNA损伤及其低效BER与CAG重复有关 扩张.该假设将通过两个具体目标进行探讨。目的一是确定环境致癌物铬酸盐和溴酸盐诱导的氧化性单链DNA（ssDNA）断裂中间体的低效加工如何参与CAG重复扩增。ssDNA断裂的不充分加工对CAG重复扩增的影响将在Pol p dRP裂解酶和发夹结构的FEN 1切割缺陷下确定。目的二是确定氧化ssDNA断裂的高效加工是否可以防止CAG重复扩增。这一目标将通过确定是否 CAG重复扩增可以通过APE 1和Pol p、XRCC 1和Pol 3以及BLM和FEN 1之间的BER蛋白相互作用有效加工ssDNA断裂中间体来减少。在威尔逊博士的指导下，我在获奖期间的指导阶段成功地实现了我的研究和职业目标。我已经建立了几种分析CAG重复不稳定性的方法， 体内和体外。这提高了我在分析体内TNR稳定性和BER生物化学方面的技能和知识。这也巩固了在独立阶段实现研究目标的基础，以及我未来R 01提案的构建。我未来的研究将侧重于环境氧化DNA损伤诱导的重复序列不稳定性的细胞和生物化学研究领域。BER突变对三核苷酸重复稳定性和BER蛋白生物化学的影响。

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.