Collaborative Proposal: Elucidating Chemical Mechanisms of DNA Repair Using Transition State Analogs

由于细胞新陈代谢和环境暴露对DNA碱基的破坏，生命DNA蓝图中持有的信息可能会被侵蚀。DNA碱基的修饰会导致DNA序列的变化，这种变化称为突变。这些突变可能会对细胞过程产生负面影响，但它们可以通过DNA修复酶的作用得到纠正。该奖项将资助加州大学戴维斯分校的Sheila David博士和犹他大学的Martin Horvath博士，以揭示DNA碱基受损检测和修复的分子细节。这项研究利用合成DNA化学和X射线结晶学来捕捉和可视化DNA修复过程中的步骤，否则这些步骤太过短暂，无法研究，并生成DNA修复过程的详细分子图像。研究生和本科生学习如何结合不同的化学和生物学方法来揭示和理解基本生命过程的关键特征。该项目还融入了加州大学戴维斯分校和犹他大学教授的独特的“以课程为基础的研究经验”(CURE)实验室课程。本科生从这门课程中获得了更好的批判性思维技能，并更好地掌握了对生命有机体至关重要的信息是如何在他们的DNA中编码并由DNA修复酶维持的概念性知识。本研究项目描述了碱基切除修复(BER)糖基酶的化学机制，以阐明这些酶如何有效地找到罕见的修饰DNA碱基。通过合成化学方法制备了模拟过渡态的修饰核苷酸。对几种与含有这些过渡态模拟物的DNA结合的BER糖基酶的结构研究为了解碱基切除的催化作用提供了线索。事实上，在三种BER糖基酶，即MutY、AlkD和MBD4中发现的令人惊讶的特征，对这些和相关的糖基酶提出了新的问题，并促使新的创新结构和功能研究，将由David和Horvath研究小组进行。从事这个项目的学生接受跨学科的培训，包括合成核酸化学、核酸酶学和DNA-蛋白质结晶学。培育妇女和少数族裔学生茁壮成长的环境是该奖项支持的工作的核心目标。这个项目具有积极的更广泛的教育影响，因为它与两门本科实验室课程捆绑在一起，这两门课程向即将入学的和高级本科生介绍化学生物学中使用的现代研究技术和方法，重点是DNA损伤和修复。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The information held within the DNA blueprint for life can be eroded by damage to DNA bases due to cellular metabolism and environmental exposures. Modified DNA bases lead to changes in the DNA sequence called mutations. These mutations can negatively impact cellular processes but they can be corrected by the action of DNA repair enzymes. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Sheila David at the University of California Davis and Dr. Martin Horvath at the University of Utah to uncover molecular details of how damaged DNA bases are detected and repaired. The research makes uses of synthetic DNA chemistry and X-ray crystallography to trap and visualize steps in the DNA repair process that are otherwise too fleeting to study and to generate a detailed molecular picture of the DNA repair process. Graduate and undergraduate students learn how to combine different chemistry and biology approaches to reveal and understand key features of fundamental life processes. This project is also integrated into a unique "Course-Based Research Experience" (CURE) laboratory course taught at both the University of California Davis and the University of Utah. Undergraduates emerge from the course with improved critical thinking skills and a better conceptual knowledge of how information crucial for living organisms is encoded in their DNA and maintained by DNA repair enzymes. This research project delineates chemical mechanisms of base excision repair (BER) glycosylases to illuminate how these enzymes efficiently find rare modified DNA bases. Modified nucleotides that mimic transition states are prepared through synthetic chemistry. Structural studies of several BER glycosylases bound to DNA containing these transition state mimics provide insight into the catalysis of base excision. Indeed, surprising features uncovered with three BER glycosylases, namely MutY, AlkD and MBD4, raise new questions on these and related glycosylases and prompt new innovative structural and functional studies that will be pursued by the David and Horvath research groups. Students working on this project receive interdisciplinary training spanning synthetic nucleic acid chemistry, nucleic acid enzymology, and DNA-protein crystallography. Fostering environments in which women and minority students thrive is a core goal of the work supported by this award. This project has a positive broader educational impact because it is tied to two undergraduate laboratory courses that introduce incoming and advanced undergraduate students to modern research techniques and approaches used in chemical biology with a focus on DNA damage and repair.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.