CAREER: Mechanisms of G4 DNA Induced Genome Instability

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。大多数DNA以双螺旋的形状存在，然而，一些DNA序列可以采用其他形状，如G四重体（G4），它类似于螺旋中的缠结。由于其不寻常的形状，G4 DNA在正常的细胞过程中起着重要的作用，例如标记基因的表达。然而，解开G4结构以进入的过程很困难，并且可能导致DNA断裂。断裂的DNA经常发生突变，有时会在细胞核外错误的细胞区室中结束；这两种结果都可能对细胞有害。该项目将研究保护G4 DNA免受断裂、突变和异常定位的机制。该研究将为本科生和研究生提供培训机会。此外，该项目将作为一个平台，吸引和留住代表性不足的少数民族学生，并在与新闻学院合作教授的课程中促进关键科学传播技能的发展。美国救援计划对该项目的资助为这位研究者在其职业生涯的关键阶段提供了支持。G4 DNA是一种非规范的DNA结构，很容易形成重复的、富含鸟嘌呤的序列。虽然G4 DNA在转录和DNA复制中具有生理作用，但它也会引发基因组不稳定。尽管G4 DNA与多种突变之间存在着充分的关联，但其潜在机制尚不完全清楚。关于细胞质中G4 DNA的异常积累，一种独特的基因组不稳定形式，所知甚少。该项目将验证特定染色质状态和内源性DNA修复过程促进G4 DNA不稳定（包括突变和错误定位）的假设。该项目将使用PRIME基因组编辑技术在人类细胞中植入亚形态突变，以及免疫荧光和靶下切割和标记（CUT&Tag）技术，对G4 DNA进行全局分析。此外，该项目将使用一种新型的基于细胞的报告细胞以及染色质免疫沉淀与下一代测序的耦合来表征G4 DNA的突变。这项工作将促进对染色质状态如何在非规范DNA结构中放大基因组不稳定性的理解。此外，这些研究将探讨导致胞质G4 DNA产生的机制，这可能构成一种由核外自由DNA片段驱动的新的信号传导模式。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。

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.