Howe Jesse, Walker Douglas, Tengler Kyle, Sonpatki Maya, Reardon Patrick N, Leung Justin W C, Barbar Elisar J
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, United States.
Department of Radiation Oncology, The University of Texas Health Science Center at San Antonio, San Antonio, United States.
Elife. 2025 Apr 29;14. doi: 10.7554/eLife.102179.
53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers. As a trimer, 53BP1 forms a heterogeneous mixture of complexes when bound to dimeric LC8 with the largest mass corresponding to a dimer-of-trimers bridged by 3 LC8 dimers. Analytical ultracentrifugation and isothermal titration calorimetry demonstrate that only the second of the three LC8 recognition motifs is necessary for a stable bridged complex. The stability of the bridged complex is tuned by multivalency, binding specificity of the second LC8 site, and the length of the linker separating the LC8 binding domain and OD. 53BP1 mutants deficient in bridged species fail to impact 53BP1 focus formation in human cell culture studies, suggesting that the primary role of LC8 is to bridge 53BP1 trimers which in turn promotes recruitment of 53BP1 at sites of DNA damage. We propose that the formation of higher-order oligomers of 53BP1 explains how LC8 elicits an improvement in 53BP1 foci and affects the structure and functions of 53BP1.
53BP1是DNA修复中的关键因子，与BRCA1共同调节DNA双链断裂修复机制的选择。53BP1将DNA修复因子定位到DNA损伤位点的过程受其寡聚化结构域（OD）以及与LC8的结合控制，LC8是一种中枢蛋白，可使100多种客户蛋白二聚化。在此，我们表明53BP1 OD是三聚体，这对于LC8的客户蛋白来说是个不寻常的发现，因为LC8的客户蛋白均为二聚体或四聚体。作为三聚体，53BP1与二聚体LC8结合时形成复合物的异质混合物，其中质量最大的对应于由3个LC8二聚体桥接的三聚体二聚体。分析超速离心和等温滴定量热法表明，三个LC8识别基序中只有第二个对于稳定的桥接复合物是必需的。桥接复合物的稳定性由多价性、第二个LC8位点的结合特异性以及分隔LC8结合结构域和OD的接头长度调节。在人类细胞培养研究中，缺乏桥接物种的53BP1突变体不会影响53BP1集落形成，这表明LC8的主要作用是桥接53BP1三聚体，进而促进53BP1在DNA损伤位点的募集。我们提出，53BP1高阶寡聚体的形成解释了LC8如何改善53BP1集落并影响53BP1的结构和功能。
