Department of Biosciences, Rice University, Houston, TX 77005, USA.
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Nucleic Acids Res. 2023 Jan 11;51(1):463-474. doi: 10.1093/nar/gkac1201.
DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.
DNA 聚合酶θ（Pol θ）在修复 DNA 双链断裂的微同源介导末端连接（MMEJ）途径中发挥着重要作用。然而，Pol θ识别微同源 DNA 末端并进行低保真度 DNA 合成的机制尚不清楚。在这里，我们展示了长达 2.4Å分辨率的尖牙鲈 Pol θ 聚合酶结构域与长和短双链 DNA 的低温电子显微镜结构。有趣的是，Pol θ 与长和短 DNA 底物的结合方式相似，在活性位点周围有广泛的相互作用。此外，Pol θ 与高保真 A 族聚合酶具有相似的活性位点，其指结构域完全闭合，但不同之处在于围绕新生碱基对存在亲水性残基。计算模拟和突变研究表明，Pol θ 的独特插入环有助于稳定短 DNA 的结合并组装 MMEJ 修复的活性位点。总之，我们的研究结果阐明了 Pol θ 介导的 MMEJ 的结构基础。
