Mechanisms of Mammalian Double-Strand Break Repair

DNA聚合酶q（Polq）是一种独特的聚合酶-解旋酶融合蛋白， 修复途径称为微同源介导的末端连接（MMEJ）或替代性末端连接。Polq函数 在通过DSB的5 '-3'核酸外切酶切除产生的3'单链DNA（ssDNA）突出端上，类似于 同源重组（HR）机制。Polq促进HR缺陷的癌细胞的生存 由于BRCA 1/2突变，并赋予对化疗剂的抗性（即电离辐射， 依托泊苷）。因此，阐明这种相对新发现的途径的分子机制是一个优先事项。 大（290 kDa）全长Polq蛋白如何执行MMEJ并在原子和分子水平上组织 水平尚不清楚。我们最近发表的体外研究表明，聚合酶和 Polq的解旋酶亚结构域对于MMEJ至关重要。其他已发表的初步研究表明， 全长Polq主要表现为单体。此外，一种新开发的GFP报告细胞测定法， 揭示了Polq独立的MMEJ机制的存在。我们计划全面阐明 Polq依赖的MMEJ，使用cryo-EM解决全长Polq的结构，并识别和表征Polq- 通过制定以下目标，建立独立的MMEJ机制：1.为了阐明Polq子域 为MMEJ做出贡献; 2.解决全长Polq的低温电镜结构; 3.调查波尔克独立党 MMEJ的机制我们希望这些研究将确定Polq和以前的新机制， 未发现的MMEJ过程可能有助于基因组不稳定性和细胞对遗传毒性的抗性 化疗药物。

DNA polymerase q (Polq) is a unique polymerase-helicase fusion protein that promotes the mutagenic DSB repair pathway called microhomology-mediated end-joining (MMEJ), or alternative end-joining. Polq functions on 3’ single-strand DNA (ssDNA) overhangs generated by 5’-3’ exonuclease resection of DSBs, similar to the homologous recombination (HR) machinery. Polq promotes the survival of cancer cells that are deficient in HR due to BRCA1/2 mutations, and confers resistance to chemotherapeutic agents (i.e. ionizing radiation, etoposide). Thus, elucidating the molecular mechanisms of this relatively newly discovered pathway is a priority. How the large (290 kDa) full-length Polq protein performs MMEJ and is organized at the atomic and molecular level remains unclear. Our recently published in vitro studies indicate that attachment of the polymerase and helicase subdomains of Polq is essential for MMEJ. Additional published preliminary studies demonstrate that full-length Polq behaves primarily as monomers. Furthermore, a newly developed GFP reporter cellular assay reveals the existence of Polq-independent MMEJ mechanisms. We plan to fully elucidate the molecular basis of Polq-dependent MMEJ, solve the structure of full-length Polq using cryo-EM, and identify and characterize Polq- independent MMEJ mechanisms by developing the following aims: 1. To elucidate how Polq subdomains contribute to MMEJ; 2. To solve the cryo-EM structure of full-length Polq; 3. To investigate Polq-independent mechanisms of MMEJ. We expect that these studies will identify novel mechanisms of Polq and previously undiscovered MMEJ processes that likely contribute to genome instability and cellular resistance to genotoxic chemotherapy agents.