DNA Repair Gene Research, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba, Japan.
Exp Cell Res. 2011 Oct 1;317(16):2267-75. doi: 10.1016/j.yexcr.2011.06.015. Epub 2011 Jul 2.
Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.
Ku 是由 Ku70 和 Ku80 组成的异二聚体，在 DNA 双链断裂（DSB）修复途径中发挥重要作用，即非同源末端连接（NHEJ）。同一基因编码的 Ku80 的两种同工型，即 Ku80 和 KARP-1，在灵长类细胞中表达和发挥功能，但在啮齿类细胞中不表达。Ku80 与 Ku70 形成异二聚体。然而，目前尚不清楚 KARP-1 是否与 Ku70 形成异二聚体并发挥作用。尽管 KARP-1 似乎在 NHEJ 中起作用，但它的生理作用仍不清楚。在本研究中，我们建立并鉴定了表达 EGFP-KARP-1 的 xrs-6 细胞系，即 EGFP-KARP-1/xrs-6。我们发现 KARP-1 的核定位信号（NLS）位于 C 端区域。我们的数据表明，KARP-1 以依赖和不依赖 NLS 的方式定位于核内，并与 Ku70 形成异二聚体，稳定 Ku70。另一方面，EGFP-KARP-1 不能完全弥补 Ku80 缺陷的 xrs-6 细胞的放射敏感性和 DSB 修复活性。此外，尽管 EGFP-KARP-1 可在 DSB 处积累，但 KARP-1 不能比 Ku80 更快地积累到 DSB 处。我们的数据表明，尽管 KARP-1 具有一些与 Ku80 相似的生化特性并与 Ku70 形成异二聚体，但它在 DSB 修复中的功能不能完全替代 Ku80。另一方面，在 X 射线照射后，表达 EGFP-KARP-1 的 xrs-6 细胞中出现全核 γ-H2AX 染色的细胞数量显著增加，这表明 KARP-1 可能在 DSB 反应中具有新的作用。
