CAREER: Molecular Recognition of 8-oxoguanine Modified G-Quadruplexes by the FANCJ Helicase and the REV1 Polymerase

职业：FANCJ 解旋酶和 REV1 聚合酶对 8-氧代鸟嘌呤修饰的 G-四链体的分子识别

• 批准号: 2142839
• 负责人: Colin Wu
• 金额: $ 94.59万
• 依托单位: Oakland University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2142839&HistoricalAwards=false
• 关键词: CAREER; Molecular; Recognition; oxoguanine; Modified

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).DNA is the “blueprint of life” that contains the instructions needed for cells to grow. That information must be duplicated accurately when a cell divides; otherwise, errors will be embedded into the DNA template and be passed onto the future generations of cells. Cellular DNA undergoes harmful modifications daily as a result of chemical and environmental stressors. The goal of this project is to determine the precise mechanisms by which repair proteins recognize and remove damaged DNA. The experiments will be incorporated into the CHM 4257 (Biochemistry Laboratory) curriculum to increase the access of basic research for Oakland University (OU) undergraduates. Since 24 students enroll in CHM 4257 each year, this project will provide research opportunities for up to 120 OU undergraduates. CHM 4257 students will gain a more authentic laboratory experience instead of performing a set of scripted procedures. To integrate research with outreach, OU students will demonstrate genetic testing (for lactose tolerance) in high school science classrooms with an all-in-one DNA analysis kit from Bento Lab. By mobilizing research, students and teachers who have limited resources will have a convenient way to use laboratory equipment. The outreach activities will allow OU undergraduates to work with high school students and teachers to reinforce textbook principles through hands-on experiences. This project will engage students in research, classroom education, and community outreach to prepare them for a future career in science. American Rescue Plan funding is used to support this early career investigator at a critical stage in his career.G-quadruplexes (G4s) are ubiquitous nucleic acid structures that are formed by guanine-rich sequences. G4s are especially prone to oxidative damage and 8-oxoguanine modified G4s (8oxoG4s) must be removed timely in dividing cells. The FANCJ helicase and REV1 polymerase have emerged as first responders in the repair of 8oxoG4s; however, the precise mechanisms by which FANCJ and REV1 assemble onto DNA to coordinate the unfolding of 8oxoG4s with translesion synthesis are not yet clear. The goals of this project are to define the fundamental role of 8oxoG in not only the thermal and mechanical stability of 8oxoG4s, but also the binding of FANCJ and REV1. The DNA binding properties of FANCJ and REV1 are expected to correlate with their repair functions in human cells. To test this working hypothesis, the impact of 8oxoG on the structure and thermal stability of 8oxoG4s will be examined by circular dichroism spectroscopy; the mechanical stability and unfolding mechanism of the DNA will be determined by single-molecule “fleezers.” Next, the affinities of FANCJ and REV1 for 8oxoG4s will be measured by biolayer interferometry. Lastly, the effects of 8oxoG4s on the DNA repair and contractile phenotypes of human heart muscle cells will be determined by modified comet assays and video-based imaging. These results will define the relationship between oxidative DNA damage and cardiomyocyte function for the first time, and they will facilitate future research to delineate the unknown phenotypes of FANCJ and REV1 variants.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.

该奖项全部或部分由2021年美国救援计划法案（公法117-2）资助。DNA是“生命蓝图”，包含细胞生长所需的指令。当细胞分裂时，这些信息必须准确地复制;否则，错误将嵌入DNA模板并传递给后代细胞。由于化学和环境压力，细胞DNA每天都在进行有害的修改。该项目的目标是确定修复蛋白识别和去除受损DNA的精确机制。这些实验将被纳入CHM 4257（生物化学实验室）课程，以增加奥克兰大学（Oakland University）本科生的基础研究机会。由于每年有24名学生报名参加CHM 4257，该项目将为多达120名本科生提供研究机会。CHM 4257学生将获得更真实的实验室体验，而不是执行一套脚本程序。为了将研究与推广相结合，EMBA学生将在高中科学教室中使用Bento Lab的一体化DNA分析工具包演示遗传测试（乳糖耐受性）。通过动员研究，资源有限的学生和教师将有一个方便的方式使用实验室设备。外展活动将使大学本科生与高中学生和教师合作，通过实践经验加强教科书的原则。该项目将让学生参与研究，课堂教育和社区推广，为他们未来的科学事业做好准备。 美国救援计划基金用于支持这位早期职业研究者在其职业生涯的关键阶段。G-四链体（G4）是由富含鸟嘌呤的序列形成的普遍存在的核酸结构。G4特别容易受到氧化损伤，并且8-氧代鸟嘌呤修饰的G4（8 oxoG 4）必须在分裂细胞中及时去除。FANCJ解旋酶和REV 1聚合酶已经成为8 oxoG 4修复的第一反应者;然而，FANCJ和REV 1组装到DNA上以协调8 oxoG 4的解折叠与跨损伤合成的精确机制尚不清楚。该项目的目标是确定8 oxoG不仅在8 oxoG 4的热稳定性和机械稳定性中，而且在FANCJ和REV 1的结合中的基本作用。FANCJ和REV 1的DNA结合特性预计与它们在人类细胞中的修复功能相关。为了验证这一工作假设，将通过圆二色光谱检查8 oxoG对8 oxoG 4s的结构和热稳定性的影响; DNA的机械稳定性和解折叠机制将通过单分子“fleezer”确定。接下来，将通过生物层干涉测量法测量FANCJ和REV 1对8 oxoG 4的亲和力。最后，8 oxoG 4s对人类心肌细胞DNA修复和收缩表型的影响将通过改良的彗星试验和基于视频的成像来确定。这些结果将首次定义氧化性DNA损伤和心肌细胞功能之间的关系，并将促进未来的研究，以描绘FANCJ和REV 1变体的未知表型。该奖项反映了NSF的法定使命，并已被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。