CAREER: Mechanisms of G4 DNA Induced Genome Instability

该奖项全部或部分根据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序列可以采用替代形状，例如G四链体（G4），其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结构的过程是困难的，可能会导致DNA断裂。断裂的DNA通常会发生突变，有时会出现在细胞核外错误的细胞区室中;这两种结果都可能对细胞有害。该项目将调查知之甚少的机制，保护G4 DNA断裂，突变和异常定位。这项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沟通技能的发展。 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，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，但它也引发基因组不稳定性。尽管G4 DNA与多种突变之间的关联有充分的证据，但其潜在机制尚未完全了解。关于G4 DNA在细胞质中的异常积累（一种独特的基因组不稳定性形式），我们知道的甚至更少。该项目将测试特定染色质状态和内源性DNA修复过程促进G4 DNA不稳定性（包括突变和错误定位）的假设。该项目将使用PRIME基因组编辑在人类细胞中安装亚型突变，以及免疫荧光和靶下切割和标签化（CUT Tag）来全球分析G4 DNA。此外，该项目将使用一种新的基于细胞的报告沿着染色质免疫沉淀耦合到下一代测序，以表征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，这些研究将调查导致细胞质G4 DNA的机制，这可能构成一个新的信号模式驱动的游离DNA片段外的nuclear.This奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

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.