Structure and Function of DNA Polymerase Theta

DNA 聚合酶 Theta 的结构和功能

• 批准号: 10094002
• 负责人: Richard T Pomerantz
• 金额: $ 38.79万
• 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/10094002
• 关键词: ATP phosphohydrolase; Address; Base Pairing; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; CRISPR/Cas technology; Chromosomal translocation; Cicatrix; Collaborations; Complex; Crystallization; DNA; DNA Double Strand Break; DNA Integration; DNA Repair; DNA annealing; DNA-Directed DNA Polymerase; Data; Double Strand Break Repair; Eukaryota; Exhibits; Family; Fishes; Genes; Genomic Instability; Growth; Human; Knock-in; Lead; Length; Malignant Neoplasms; Mammalian Cell; Mediating; Molecular; Mus; Mutation; N-terminal; Pathway interactions; Plant Genome; Plants; Plasmids; Polymerase; Proteins; RAD52 gene; Reporter; Resistance; Robotics; Role; Single-Stranded DNA; Site; Structure; T-DNA; Testing; arm; base; cancer cell; chemotherapy; collaborative approach; ds-DNA; genome editing; helicase; homologous recombination; insight; mammalian genome; novel; polymerization; recombinase; repaired; tripolyphosphate

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 DNA聚合酶θ(POLθ)是高等真核生物中的一种独特的蛋白质，因为它含有一个N末端 超家族2解旋酶结构域(POLθ-解旋酶)和C末端A家族聚合酶结构域(POLθ- 聚合酶)。POLθ是DNA双链断裂修复途径替代末端连接所必需的 (ALT-EJ)，也称为微同源介导的末端连接(MMEJ)。POLθ还促进了 癌细胞在同源定向修复(HDR)中有缺陷，并对化疗药物产生耐药性。这个 然而，POLθ活性的结构和分子基础仍不清楚。例如，POLθ的活动- 解旋酶知之甚少，而POLθ聚合酶如何独特地作用于单个- 链DNA(SsDNA)悬吊并促进MMEJ，目前尚不清楚。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-解旋酶的新功能，包括单链DNA退火和DNA解链，并具有 确定了POLθ聚合酶的最佳底物，这将使MMEJ的结晶学研究成为可能。 最后，我们发现了POLθ在CRISPR-CAS9基因组编辑中的一个意想不到的重要作用。我们 将利用三个实验室(Pmerantz，Chen和Sfeir)之间的合作方法来阐明结构 以及POLθ在DNA修复和基因组编辑中活性的分子基础，主要有以下目的：1. 研究Polθ的退火、解离和抗重组酶活性；2.阐明Pol DNA的结构基础 POLθ聚合酶依赖的MMEJ；3.研究POLθ参与CRISPR-CAS9基因组编辑 单链DNA模板。总而言之，这些研究将提供新的和重要的洞察结构和 POLθ的功能，并描述了POLθ在CRISPR-CAS9基因组编辑中意想不到的作用。