Structure and Function of DNA Polymerase Theta

项目摘要 DNA聚合酶θ（Polθ）是高等真核生物中一种独特的蛋白质， 超家族2（SF 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-解旋酶）。 聚合酶）。Polθ在DNA双链断裂（DSB）修复途径的交替末端连接中是必需的 （alt-EJ），也称为微同源介导的末端连接（MMEJ）。Polθ还促进了 在同源定向修复（HDR）中有缺陷的癌细胞，并赋予对化疗剂的抗性。的 然而，Polθ活性结构和分子基础仍不清楚。例如，Polθ- 解旋酶知之甚少，Polθ-聚合酶如何独特地作用于单- 单链DNA（ssDNA）突出并促进MMEJ仍有待阐明。此外，如何 Polθ对CRISPR-Cas9基因组编辑的影响仍然知之甚少。在初步研究中， 发现了Polθ-解旋酶的新功能，包括ssDNA退火和DNA解旋， 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。我们 将利用三个实验室（Pomerantz，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的活性的分子基础，通过发展以下目标：1.到 研究Polθ的退火、解旋和抗重组酶活性; 2.为了阐明的结构基础 Polθ-聚合酶依赖性MMEJ; 3.为了研究Polθ参与CRISPR-Cas9基因组编辑， ssDNA模板。总之，这些研究将提供新的和重要的洞察结构， 本研究旨在揭示Polθ的功能，并表征Polθ在CRISPR-Cas9基因组编辑中的意想不到的作用。

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.