CAREER: Mechanisms of G4 DNA Induced Genome Instability

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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). Most DNA exists in the shape of a double helix however, some DNA sequences can adopt alternative shapes such as a G quadruplex (G4), which resembles a tangle in the helix. Because of its unusual shape, G4 DNA plays important roles in normal cellular processes such as marking genes for expression. However, the process of untangling a G4 structure for access is difficult and can cause DNA to break. Broken DNA often becomes mutated and sometimes ends up outside of the nucleus in the wrong cellular compartment; both outcomes can be detrimental for cells. This project will investigate poorly understood mechanisms that protect G4 DNA from breakage, mutation, and aberrant localization. The research will provide training opportunities for undergraduate and graduate students. In addition, the project will serve as a platform to engage and retain under-represented minority students and to foster development of critical science communication skills in a course taught collaboratively with journalism faculty. American Rescue Plan funding of this project provides support for this investigator at a critical stage in her career.G4 DNA is a non-canonical DNA structure that forms readily in repetitive, guanine-rich sequences. While G4 DNA has physiological roles in transcription and DNA replication, it also triggers genome instability. Despite a well-documented association between G4 DNA and mutations of multiple kinds, the underlying mechanisms are incompletely understood. Even less is known regarding the aberrant accumulation of G4 DNA in the cytoplasm, a unique form of genome instability. This project will test the hypothesis that specific chromatin states and endogenous DNA repair processes promote instability, including mutations and mislocalization, of G4 DNA. The project will use PRIME genome editing to install hypomorphic mutations in human cells as well as immunofluorescence and Cleavage Under Targets and Tagmentation (CUT&Tag) to globally profile G4 DNA. Additionally, the project will use a novel cell-based reporter along with coupling of chromatin immunoprecipitation to Next Generation Sequencing to characterize mutations at G4 DNA. This work will advance understanding of how chromatin states might amplify genome instability at non-canonical DNA structures. Further, these studies will investigate the mechanisms that lead to cytosolic G4 DNA, which could constitute a new signaling paradigm driven by free DNA fragments outside the nucleus.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.