DNA Base Excision Repair in Chromatin

环境中含有许多能破坏生物体DNA的活性物质。暴露在这些反应性物质中会产生一系列DNA损伤，改变细胞的遗传蓝图，威胁到所有方面的有机体功能，以及遗传信息向下一代的忠实传递。为了保护DNA，已经进化出了多种修复途径，包括碱基切除修复（BER），其中受损的DNA碱基被移除并被未受损的DNA碱基所取代。这个项目研究了BER如何作用于被蛋白质包装成染色质（DNA在细胞内的形式）的DNA。目的是确定这种包装如何影响DNA修复，并确定细胞克服与修复染色质内隔离的DNA相关的挑战的方法。该项目将为包括女子学院本科生在内的学生提供DNA化学、蛋白质生物化学、生物物理学和分子生物学方面的培训机会。这些技能将使学生能够在研究和教育领域从事STEM职业，并为生物技术行业做出贡献。还计划对中学生进行科学宣传。BER是一系列协调的酶催化化学反应，其中DNA中受损的碱基被移除和替换。这一基本细胞过程的矛盾之处在于，当DNA被包装在染色质中时，BER修复DNA的能力仍然知之甚少，这可以限制酶接近受损的碱基。这项研究将采用一种创新的“修复指纹”技术，该技术可以精确识别染色质中BER酶具有催化活性的位置以及难以修复的位置。该技术以及各种生物化学、生物物理学和分子生物学方法将被用于(i)确定包装DNA中DNA碱基翻转是如何控制的，（ii）了解DNA包装密度如何调节BER，以及（iii）确定包装DNA中损伤传感器蛋白对BER的贡献。这些结果将有助于回答有关细胞机制的关键问题，以克服包装悖论并防止基因组不稳定。这项研究由生物科学理事会分子和细胞生物科学部的遗传机制项目资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The environment contains many reactive species that can damage DNA in living organisms. Exposure to these reactive species generates an array of DNA damage lesions that alter the genetic blueprint of cells and threaten all aspects organismal function, as well as the faithful transfer of genetic information to the next generation. A variety of repair pathways have evolved to protect DNA, including base excision repair (BER) in which damaged DNA bases are removed and replaced with undamaged ones. This project investigates how BER acts on DNA packaged by proteins into chromatin – the form DNA takes inside cells. The goal is to determine how this packaging influences DNA repair and to identify ways that cells overcome challenges associated with repairing DNA secluded within chromatin. The project will offer training opportunities to students, including undergraduates from women’s colleges, in DNA chemistry, protein biochemistry, biophysics, and molecular biology. These skills will enable students to pursue STEM careers in research and education, and to contribute to biotechnology industries. Science outreach to middle school students is planned as well.BER is a coordinated series of enzyme-catalyzed chemical reactions in which a damaged base in DNA is removed and replaced. A paradoxical aspect of this fundamental cellular process that still remains poorly understood is the ability of BER to repair DNA when it is packaged in chromatin, which can limit enzyme access to damaged bases. The research will employ an innovative “repair fingerprint” technique that can precisely identify locations in chromatin where BER enzymes are catalytically active as well as those that are refractory to repair. This technique, as well as a variety of biochemistry, biophysics, and molecular biology approaches will be employed to (i) determine how DNA base flipping is controlled in packaged DNA, (ii) understand how the density of DNA packaging modulates BER, and (iii) establish the contribution of a damage sensor protein to BER in packaged DNA. The outcomes will help answer key questions regarding cellular mechanisms to overcome the packaging paradox and prevent genomic instability.This research is funded by the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.