DNA glycosylases involved in interstrand crosslink repair and antibiotic self-resistance

DNA通过与细胞和环境中的分子相互作用而发生化学改变或破坏。所有生物体都含有修复受损DNA的酶，以保护遗传信息的完整性。这个研究项目将确定在细菌中发现的一种新发现的DNA修复酶是如何修复由某些细菌产生的防御机制的特定毒素造成的损伤的。由于它们的毒性，这些细菌衍生的毒素通常具有抗菌和抗肿瘤的特性，使它们在农业、工业和医学中很重要。该项目将通过以下方式造福社会：1)为本科生和研究生提供用于确定蛋白质原子结构的方法的实践经验，这些方法对生物技术的发现和创新至关重要；2)为菲斯克大学（Fisk University）的本科生提供夏季研究机会，菲斯克大学是附近的一所历史悠久的黑人学院/大学；3)为所有学员提供教学和指导机会；4)让对STEM感兴趣的高中生接触生物医学研究。5)社区拓展。这些活动是通过范德比尔特大学文理学院和医学院交叉的协作和包容性培训环境来促进的。链间DNA交联（ICLs）是最具细胞毒性的DNA损伤形式之一，因为它们以共价连接两条DNA链，从而干扰DNA复制和转录。最近在真核生物和原核生物中发现了一种新的ICL修复途径，其中DNA糖基酶从DNA主链中释放一个交联的核碱基。细菌ICL糖基酶属于抗生素生产者和病原体中普遍存在的未表征蛋白质家族。PI的实验室发现，大肠杆菌YcaQ通过解钩化学上不同的ICL来启动ICL修复途径，并表征了一种相关酶（AlkZ），该酶通过解钩产生的azinomycin B-ICL，为链霉菌提供了对其天然产物之一azinomycin B毒性的自我抗性。该项目的长期目标是阐明细菌中的ICL修复途径，了解微生物如何利用DNA修复来保护自然界和微生物组内的遗传毒性天然产物，并发现新的有益应用的遗传毒性药物。短期目标是了解YcaQ解钩ICL的分子基础，以及alkz相关酶对高功能化交联和嵌入天然产物的特异性，并表征推定的基因毒素的生物活性。将采用多学科方法整合结构生物学、生物化学、遗传学、细胞生物学和代谢组学来实现这些目标。该项目由分子和细胞生物科学部/生物科学部的遗传机制项目和化学部/数学和物理科学部的生命过程化学项目支持。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。

DNA is chemically altered, or damaged, by its interaction with molecules found in the cell and in the environment. All organisms contain enzymes that repair damaged DNA to protect the integrity of the genetic information. This research project will determine how a newly discovered DNA repair enzyme found in bacteria works to repair damage created by specific toxins, which some bacteria produce as defense mechanisms. Because of their toxicity, these bacteria-derived toxins often have antimicrobial and antitumor properties, making them important in agriculture, industry, and medicine. The project will benefit society by providing 1) undergraduate and graduate students with hands-on experience in methods used to determine atomic structures of proteins that are important for discovery and innovation in biotechnology, 2) summer research opportunities to undergraduates of Fisk University, a nearby historically black college/university, 3) teaching and mentoring opportunities for all trainees, 4) exposure of high school students interested in STEM to biomedical research, and 5) community outreach. These activities are facilitated through a collaborative and inclusive training environment at the intersection of the College of Arts and Science and the School of Medicine at Vanderbilt University.Interstrand DNA crosslinks (ICLs) are among the most cytotoxic forms of DNA damage because they covalently tether the two DNA strands and therefore interfere with DNA replication and transcription. A new ICL repair pathway was recently discovered in both eukaryotes and prokaryotes, in which a DNA glycosylase liberates one of the crosslinked nucleobases from the DNA backbone. The bacterial ICL glycosylases belong to a family of uncharacterized proteins prevalent in antibiotic producers and pathogens. The PI’s laboratory discovered that E. coli YcaQ initiates an ICL repair pathway by unhooking chemically diverse ICLs, and characterized a related enzyme (AlkZ) that provides Streptomyces with self-resistance to the toxicity of one of its natural products, azinomycin B, by unhooking resulting azinomycin B-ICLs. The long-term goals of this project are to elucidate the ICL repair pathway in bacteria, to understand how microbes utilize DNA repair to protect against genotoxic natural products in nature and within the microbiome, and to discover new genotoxic agents with beneficial applications. The short-term goals are to understand the molecular basis for ICL unhooking by YcaQ and for the specificity of AlkZ-related enzymes for highly functionalized crosslinking and intercalating natural products, and to characterize the bioactivities of putative genotoxins. A multidisciplinary approach integrating structural biology, biochemistry, genetics, cell biology, and metabolomics will be employed to achieve these goals.This project is supported by the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences/Directorate for Biological Sciences and the Chemistry of Life Processes program in the Division of Chemistry/Directorate for Mathematical and Physical 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.