Structure and Function of DNA Polymerase Theta

DNA 聚合酶 Theta 的结构和功能

基本信息

批准号：
10377900
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-15 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10377900
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

项目摘要

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.

项目概要 DNA 聚合酶 θ (Polθ) 是高等真核生物中一种独特的蛋白质，因为它含有一个 N 末端超家族 2 (SF2) 解旋酶结构域 (Polθ-解旋酶) 和 C 端 A 家族聚合酶结构域 (Polθ- Polθ 对于 DNA 双链断裂 (DSB) 修复途径替代末端连接至关重要。（alt-EJ），也称为微同源介导的末端连接（MMEJ）也促进增殖。癌细胞在同源定向修复（HDR）方面存在缺陷，并对化疗药物产生耐药性。然而，Polθ 活性的结构和分子基础仍不清楚，例如 Polθ- 的活性。人们对解旋酶知之甚少，并且 Polθ-聚合酶如何独特地作用于单链的结构基础 DNA 链 (ssDNA) 突出并促进 MMEJ 此外，如何发挥作用仍有待阐明。在初步研究中，我们对 Polθ 对 CRISPR-Cas9 基因组编辑的影响仍知之甚少。发现了 Polθ-解旋酶的新功能，包括 ssDNA 退火和 DNA 解旋，并已确定了 Polθ 聚合酶 MMEJ 的最佳底物，这将使 MMEJ 的晶体学研究成为可能。最后，我们发现了 Polθ 在 CRISPR-Cas9 基因组编辑中意想不到的重要作用。将利用三个实验室（Pomerantz、Chen 和 Sfeir）之间的合作方法来阐明结构通过制定以下目标，研究 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、解旋和抗重组酶活性2.阐明其结构基础； 3. 使用 Polθ 聚合酶依赖的 MMEJ 研究 Polθ 参与 CRISPR-Cas9 基因组编辑的情况总之，这些研究将为结构和结构提供新的、重要的见解。 Polθ 的功能，并描述了 Polθ 在 CRISPR-Cas9 基因组编辑中的意想不到的作用。