Suppression of DNA-damage dependent total chromatid premature separation by p53-binding protein 1

DNA损伤后，p53结合蛋白1（53 BP 1）重新定位到离散的核灶，表明其在DNA损伤反应中的作用。我们已经鉴定了结肠癌细胞系SW 48缺乏53 BP 1表达，并建立了以强力霉素抑制的方式表达53 BP 1的细胞系SW 48 dox 53 BP 1。当用X射线照射53 BP 1非表达细胞时，它们显示出完全过早染色单体分离（PCS），这是一种即使细胞处于中期也显示出姐妹染色单体分离的表型。在53 BP 1表达细胞中未观察到PCS表型，表明53 BP 1调节X射线照射的细胞中的姐妹染色单体分离。53 BP 1特异性刺激DNA连接酶IV/Xrcc 4的末端连接，表明53 BP 1直接参与DNA双链断裂（DSB）的非同源末端连接（NHEJ）修复。我们使用53 BP 1敲除的鸡DT 40细胞进一步研究了53 BP 1在DNA DSB修复中的功能，发现53 BP 1参与了另一种DSB修复途径，与经典的NHEJ不同，该途径对渥曼青霉素具有抗性，并且优先在细胞周期的G1期发挥作用。

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to discrete nuclear foci, suggesting its role in DNA damage responses. We have identified that a colon cancer cell line SW48 is deficient in 53BP1 expression and established a cell line, SW48dox53BP1, which expresses 53BP1 in a doxycycline-repressible manner. When 53BP1 non-expressing cells were irradiated with X-ray, they showed total premature chromatid separation (PCS), a phenotype that shows sister chromatid separation even though cells are in the metaphase. The PCS phenotype was not observed in 53BP1 expressing cells, suggesting that 53BP1 regulates sister chromatid separation in cells irradiated with X-ray. 53BP1 specifically stimulated end-joining by DNA ligase IV/Xrcc4, suggesting that 53BP1 directly participates in the non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). We further investigated 53BP1 functions in DNA DSB repair using 53BP1-knockout chicken DT40 cells, and found that 53BP1 is involved in an alternative DSB repair pathway that, unlike the classical NHEJ, is resistant to wortmannin and functions preferentially in the G1 phase of the cell cycle.