Mechanism and Specificity of DNA Repair Dealkylases

基因组DNA经历了自发的化学修饰(烷基化)，细胞或环境中的活性物质改变了它的结构和信息含量；因此，细胞需要DNA修复途径来检测和抵消这些有害的变化。这项研究将表征一种新发现的称为直接修复的途径的机制和特异性，在这种途径中，DNA修复脱烷基酶通过涉及多个步骤的反应恢复正确的核苷酸。通过将各级受训人员纳入该项目，PI将使个人为未来在STEM相关领域的职业生涯做好准备。学生将接受遗传、生化和生物物理技术方面的跨学科培训，同时获得蛋白质和核酸生物化学方面的专门知识。该项目将继续关注从代表性不足的群体中招收学生，并向社区推广，从小学到医学院，以激发人们对基础科学的热情，并培养多样化的科学社区。该项目确定ALKB和ALKBH2是DNA修复氧化脱烷酶超家族的代表，并解决了一些重要的悬而未决的问题，如这些酶如何搜索DNA中的损伤，区分修复底物和非底物，以及保持对补充其他已知DNA修复途径的广泛底物的特异性。中心假设是，ALKB酶的机制与独立进化的DNA糖基酶有相似之处，后者使用不同的化学和不同的结构折叠来克服相似的生物和物理挑战。这项研究将应用酶学、瞬时动力学、结构/功能分析、动力学建模和遗传学相结合的方法来了解ALKB和ALKBH2发现和修复DNA损伤的机制。该项目由生物科学局分子和细胞生物科学部的遗传机制小组和数学和物理科学局化学部的生命过程化学共同资助。

Genomic DNA undergoes spontaneous chemical modification (alkylation) whereby cellular or environmental reactive chemicals alter its structure and information content; therefore, cells require DNA repair pathways to detect and counteract these deleterious changes. This research will characterize the mechanism and specificity of a newly discovered pathway called direct repair in which a DNA repair dealkylase restores the correct nucleotide via a reaction involving multiple steps. By integrating trainees at all levels into the project, the PI will prepare individuals for future careers in STEM-related fields. Students will receive interdisciplinary training in genetic, biochemical, and biophysical techniques while gaining specific expertise in protein and nucleic acid biochemistry. A continued focus will be on recruiting students from underrepresented groups and outreach to the community, from elementary school all the way up to medical school, to incite a passion for basic sciences and to foster a diverse scientific community.The project identifies ALKB and ALKBH2 as representative examples of a broad superfamily of DNA repair oxidative dealkylases and addresses important unanswered questions about how these enzymes search for lesions in DNA, distinguish repair substrates from non-substrates, and maintain specificity for a broad range of substrates that complement other known DNA repair pathways. The central hypothesis is that the mechanism of ALKB enzymes has parallels to independently evolved DNA glycosylases that use different chemistry and different structural folds to overcome similar biological and physical challenges. The research will apply a combined approach of enzymology, transient kinetics, structure/function analysis, kinetic modeling, and genetics to understand the mechanisms by which ALKB and ALKBH2 find and repair DNA damage.This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate for Mathematical and Physical Sciences.