Department of Pathology, Department of Biochemistry & Molecular Biology, Department of Molecular Microbiology & Immunology, and Section of Computational & Molecular Biology, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1441 Eastlake Ave, Rm. 5428, Los Angeles, CA 90089, USA.
Eyring Materials Center, John Cowley Center for High Resolution Electron Microscopy, Arizona State University, Tempe, AZ 85281, USA.
Nucleic Acids Res. 2022 Jul 22;50(13):7697-7720. doi: 10.1093/nar/gkac564.
Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form (basal state) of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation.
Artemis 核酸酶和 DNA 依赖性蛋白激酶催化亚基（DNA-PKcs）是非同源末端连接（NHEJ）的关键组成部分，NHEJ 是双链 DNA 断裂的主要修复机制。DNA-PKcs 对 Artemis 的激活可解决 V(D)J 重组过程中形成的发夹 DNA 末端。Artemis 缺陷会破坏适应性免疫的发育，并导致对辐射敏感的 T-B-严重联合免疫缺陷（RS-SCID）。最近通过 cryo-EM 解决了 Artemis 与 DNA-PK 形成的激活状态，结果表明 Artemis 与 DNA 结合。在这里，我们报告说 Artemis:DNA-PKcs 复合物的预激活形式（基础状态）在琼脂糖-丙烯酰胺凝胶系统中稳定，适合 cryo-EM 结构分析。结构表明，在 ABCDE 自身磷酸化之前，Artemis 催化结构域动态定位在 DNA-PKcs 外部，并显示 Artemis 的催化和调节结构域如何与 DNA-PKcs 的 N-HEAT 和 FAT 结构域相互作用。我们定义了 DNA-PKcs 上 Artemis 和 XRCC4 的互斥结合位点，并表明 XRCC4 肽会破坏 Artemis:DNA-PKcs 复合物。所有的发现都有助于解释 DNA-PKcs 的 L3062R 错义突变如何导致 Artemis 激活不足，从而导致 RS-SCID。我们的结果提供了各种靶位点候选物，用于设计破坏 Artemis:DNA-PKcs 复合物形成的抑制剂。
