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.

项目总结