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

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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.