Frit Philippe, Amin Himani, Zahid Sayma, Barboule Nadia, Hall Chloe, Matharu Gurdip, Hardwick Steven W, Chauvat Jeanne, Britton Sébastien, Chirgadze Dima Y, Ropars Virginie, Charbonnier Jean-Baptiste, Calsou Patrick, Chaplin Amanda K
Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III-Paul Sabatier (UT3), Toulouse, France.
Leicester Institute for Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, UK.
Nat Commun. 2025 May 6;16(1):4208. doi: 10.1038/s41467-025-59133-2.
Non-homologous end joining (NHEJ) is the main repair pathway for double-strand DNA breaks (DSBs) in mammals. DNA polymerases lambda (Pol λ) and mu (Pol μ), members of the Pol X family, play a key role in this process. However, their interaction within the NHEJ complexes is unclear. Here, we present cryo-EM structures of Pol λ in complex with the DNA-PK long-range synaptic complex, and Pol μ bound to Ku70/80-DNA. These structures identify interaction sites between Ku70/80 and Pol X BRCT domains. Using mutants at the proteins interface in functional assays including cell transfection with an original gap-filling reporter, we define the role of the BRCT domain in the recruitment and activity of the two Pol X members in NHEJ and in their contribution to cell survival following DSBs. Finally, we propose a unified model for the interaction of all Pol X members with Ku70/80.
非同源末端连接（NHEJ）是哺乳动物双链DNA断裂（DSB）的主要修复途径。DNA聚合酶λ（Pol λ）和μ（Pol μ）属于Pol X家族成员，在这一过程中起关键作用。然而，它们在NHEJ复合物中的相互作用尚不清楚。在此，我们展示了与DNA-PK长程突触复合物结合的Pol λ以及与Ku70/80-DNA结合的Pol μ的冷冻电镜结构。这些结构确定了Ku70/80与Pol X BRCT结构域之间的相互作用位点。通过在蛋白质界面处使用突变体进行功能测定，包括用原始的缺口填充报告基因进行细胞转染，我们确定了BRCT结构域在NHEJ中两种Pol X成员的募集和活性以及它们对DSB后细胞存活的贡献中的作用。最后，我们提出了一个关于所有Pol X成员与Ku70/80相互作用的统一模型。
