"DNA Binding Region" of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint.
"DNA Binding Region" of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint.
复制标题
DOI：
10.7150/ijbs.14242
复制
发表时间：
2016
影响因子：
9.2
通讯作者：
Deng CX
中科院分区：
文献类型：
--
作者：
Masuda T;Xu X;Dimitriadis EK;Lahusen T;Deng CX
乳腺癌相关基因 1 (BRCA1) 包含 3 个结构域：具有泛素 E3 连接酶活性的 N 端 RING 结构域、C 端 BRCT 蛋白相互作用结构域和中心区域。 RING 和 BRCT 结构域已得到很好的表征，但中心区域的功能仍不清楚。在这项研究中，我们在人类 BRCA1 的中心区域内鉴定了一个必需的 DNA 结合区域（DBR：421-701 个氨基酸），并发现 BRCA1 将 DNA 聚集在一起，并优选以序列无关的方式与张开臂 DNA 结合。为了研究 DBR 的生物学作用，我们使用 TALEN 方法生成了缺乏 DBR (ΔDBR) 的小鼠 ES 细胞。与用 PARP 抑制剂处理的野生型 (WT) 细胞相比，ΔDBR 细胞的存活率降低，但它们具有完整的能力，可以通过同源重组 (HR) 介导进行 DNA 修复。在羟基脲 (HU) 存在的情况下，ΔDBR 细胞继续掺入更多的 EdU，这会导致复制应激并表现出比 WT 细胞更低的生存能力。此外，调节 S 期内检查点的 CHK1 磷酸化在 ΔDBR 细胞中适度降低。这些数据表明，BRCA1 与 DNA 的结合会影响 DNA 复制过程的稳定性，导致 ΔDBR 细胞中 S 期内检查点控制减弱。与WT细胞相比，ΔDBR细胞还表现出更多的异常染色体结构，表明ΔDBR细胞的遗传不稳定性增加。因此，我们证明了 BRCA1 的 DBR 通过复制应激激活的 S 期内检查点来调节遗传稳定性。
The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress.