Structure and Function of DNA Polymerase Theta

DNA 聚合酶 Theta 的结构和功能

• 批准号: 10336827
• 负责人: Richard T Pomerantz
• 金额: $ 11.34万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-15 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10336827
• 关键词: Structure; Function; DNA; Polymerase; Theta

Project summary DNA polymerase θ (Polθ) is a unique protein in higher eukaryotes because it contains a N-terminal superfamily 2 (SF2) helicase domain (Polθ-helicase) and a C-terminal A-family polymerase domain (Polθ- polymerase). Polθ is essential for the DNA double-strand break (DSB) repair pathway alternative end-joining (alt-EJ), also called microhomology-mediated end-joining (MMEJ). Polθ also promotes the proliferation of cancer cells defective in homology-directed repair (HDR) and confers resistance to chemotherapy agents. The structural and molecular basis of Polθ activity, however, remains unclear. For example, the activities of Polθ- helicase are poorly understood, and the structural basis for how Polθ-polymerase uniquely acts on single- strand DNA (ssDNA) overhangs and promotes MMEJ remains to be elucidated. Furthermore, how Polθ influences CRISPR-Cas9 genome editing remains poorly understood. In preliminary studies, we have discovered novel functions for Polθ-helicase including ssDNA annealing and DNA unwinding, and have identified optimal substrates for Polθ-polymerase MMEJ which will enable crystallographic studies of MMEJ. Lastly, we have discovered an unexpected and essential role for Polθ in CRISPR-Cas9 genome editing. We will utilize a collaborative approach among three labs (Pomerantz, Chen and Sfeir) to elucidate the structural and molecular basis of Polθ activity in DNA repair and genome editing by developing the following aims: 1. To investigate annealing, unwinding and anti-recombinase activities of Polθ; 2. To elucidate the structural basis of Polθ-polymerase dependent MMEJ; 3. To investigate Polθ involvement in CRISPR-Cas9 genome editing using ssDNA templates. In summary, these studies will provide new and significant insight into the structure and function of Polθ, and characterize an unexpected role for Polθ in CRISPR-Cas9 genome editing.

项目摘要 DNA聚合酶θ（polθ）是较高真核生物中的独特蛋白质，因为它包含N末端 超家族2（SF2）解旋酶结构域（polθ-酶）和C末端A-家庭聚合酶结构域（POLθ- 聚合酶）。 polθ对于DNA双链断裂（DSB）维修途径替代端连接至关重要 （alt-ej），也称为微学介导的末端加入（MMEJ）。波兰还促进了 癌细胞在同源指导修复（HDR）中有缺陷，并赋予对化学疗法剂的耐药性。 然而，polθ活性的结构和分子基础尚不清楚。例如，Polθ-的活动 解旋酶的理解很少，以及polθ-聚合酶如何唯一作用于单一 - 单位的结构基础 链DNA（ssDNA）悬垂并促进mmej仍有待阐明。此外，如何 POLθ影响CRISPR-CAS9基因组编辑仍然知之甚少。在初步研究中，我们有 发现了Polθ-螺旋酶的新功能，包括ssDNA退火和DNA，并具有 确定了POLθ-聚合酶MMEJ的最佳底物，该基材将启用MMEJ的晶体学研究。 最后，我们在CRISPR-CAS9基因组编辑中发现了Polθ的意外和重要作用。我们 将在三个实验室（Pomerantz，Chen和Sfeir）中使用协作方法来阐明结构 通过开发以下目的，在DNA修复和基因组编辑中polθ活性的分子基础：1。 研究POLθ的退火，放松和抗重组酶活性； 2。阐明 polθ-聚合酶依赖性mmej; 3。研究使用Polθ参与使用CRISPR-CAS9基因组编辑 ssDNA模板。总而言之，这些研究将为结构和 POLθ的功能，并表征了Polθ在CRISPR-CAS9基因组编辑中的意外作用。