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.

该奖项的全部或部分资金根据《2021 年美国救援计划法案》（公法 117-2）提供。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，错误将被嵌入 DNA 模板中并传递给下一代细胞。由于化学和环境压力，细胞 DNA 每天都会发生有害的修饰。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）课程中，以增加奥克兰大学（OU）本科生进行基础研究的机会。由于每年有 24 名学生注册 CHM 4257，该项目将为多达 120 名 OU 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，而不是执行一组脚本程序。为了将研究与推广结合起来，俄勒冈大学的学生将使用 Bento Lab 的一体化 DNA 分析套件在高中科学课堂上演示基因测试（乳糖耐量）。通过动员研究，资源有限的学生和教师将能够方便地使用实验室设备。外展活动将使俄勒冈大学本科生能够与高中生和教师合作，通过实践经验强化教科书原则。该项目将让学生参与研究、课堂教育和社区外展，为他们未来的科学职业做好准备。 美国救援计划的资金用于支持这位处于职业生涯关键阶段的早期职业调查员。G-四链体 (G4) 是普遍存在的核酸结构，由富含鸟嘌呤的序列形成。 G4 特别容易受到氧化损伤，必须在分裂细胞中及时去除 8-氧代鸟嘌呤修饰的 G4 (8oxoG4)。 FANCJ 解旋酶和 REV1 聚合酶已成为 8oxoG4 修复的第一响应者；然而，FANCJ 和 REV1 组装到 DNA 上以协调 8oxoG4 的展开与跨损伤合成的精确机制尚不清楚。该项目的目标是明确 8oxoG 的基本作用，不仅在 8oxoG4 的热稳定性和机械稳定性方面，而且在 FANCJ 和 REV1 的结合方面。 FANCJ 和 REV1 的 DNA 结合特性预计与其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 8oxoG 对 8oxoG4s 的结构和热稳定性的影响； DNA 的机械稳定性和展开机制将由单分子“fleezer”决定。接下来，将通过生物层干涉测量法测量 FANCJ 和 REV1 对 8oxoG4 的亲和力。最后，8oxoG4 对人类心肌细胞 DNA 修复和收缩表型的影响将通过改进的彗星测定和基于视频的成像来确定。这些结果将首次定义氧化DNA损伤与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REV1变体的未知表型。该奖项反映了NSF的法定使命，并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，被认为值得支持。