Laboratory for Advanced Nuclear Energy, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Department of Radiological Technology, Mahidol University, Nakhon Pathom 73170, Thailand.
J Radiat Res. 2021 May 12;62(3):380-389. doi: 10.1093/jrr/rrab016.
Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4's DSB repair function in normal development.
非同源末端连接是 DNA 双链断裂 (DSB) 修复的主要途径之一，也参与免疫系统中的 V(D)J 重组。因此，非同源末端连接 (NHEJ) 蛋白的突变被发现与人类和模型动物的免疫缺陷有关。已有报道称，XRCC4 突变的几位人类患者表现出小头畸形和生长缺陷，但出乎意料的是，他们的免疫功能正常。在这里，为了评估 XRCC4 这些与疾病相关的突变在放射敏感性方面的功能，我们在 XRCC4 缺陷型 M10 细胞中生成表达这些突变体的稳定转染子，并通过集落形成试验测量它们的放射敏感性。V83_S105del、R225X 和 D254Mfs68 的表达水平与野生型 XRCC4 相似，而 W43R、R161Q 和 R275X 的表达水平甚至高于野生型 XRCC4。这些突变体的转染子中 DNA 连接酶 IV 的表达水平与野生型 XRCC4 转染子相当。V83S_S105del 转染子，以及在较小程度上，D254Mfs68 转染子，与野生型 XRCC4 转染子相比，显示出显著增加的放射敏感性。W43R、R161Q、R225X 和 R275X 转染子与野生型 XRCC4 转染子相比，放射敏感性略有但具有统计学意义的增加。当与绿色荧光蛋白 (GFP) 融合表达时，R225X、R275X 和 D254Mfs*68 定位于细胞质，而其他突变体定位于细胞核。这些结果共同表明，患者 XRCC4 的缺陷可能主要归因于蛋白质数量的不足和功能受损，突显了 XRCC4 在正常发育中 DSB 修复功能的重要性。
