Department of Radiation Oncology, The University of Chicago, 5801 South Ellis Avenue, Chicago, IL, 60637, USA.
Department of Medicine, Tulane University, 1430 Tulane Avenue, New Orleans, LA, 70112, USA.
Cell Biol Int. 2018 Jan;42(1):34-44. doi: 10.1002/cbin.10846. Epub 2017 Oct 25.
BRCA1 plays a central role in DNA repair. Although N-terminal RING and C-terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B-lymphocyte cell line DT40. The combination of "homologous recombineering" and "RT-cassette" enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock-in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2-9 BRCA1 (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1 decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1 within DNA-binding region, even the conserved 7-amino acid deletion BRCA1 within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage-induced Rad 51 foci formation in human BRCA1 knock-in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.
BRCA1 在 DNA 修复中起着核心作用。尽管 N 端 RING 和 C 端 BRCT 结构域已经得到了很好的研究，但 BRCA1 中心区域的功能仍知之甚少。在这里，我们报告了 BRCA1 等位基因和功能性人 BRCA1 在鸡 B 淋巴细胞系 DT40 中的结构和功能分析。“同源重组”和“RT 盒”的结合使我们能够在大肠杆菌中对鸡 BRCA1 基因进行修饰。通过同源重组，使用带有 BRCA1 突变构建体的突变 BRCA1 敲入 DT40 细胞系的生成效率高达 100%。我们的研究表明，删除 BRCA1 中的motif 2-9（模拟秀丽隐杆线虫 BRCA1 的结构域）或删除 motif 1 BRCA1 会降低顺铂处理后的细胞活力。此外，即使是删除 motif 6 中保守的 7 个氨基酸的 BRCA1，也会导致 DNA 结合区域中的 motif 5 和 6 的缺失，从而降低顺铂处理后的细胞活力。令人惊讶的是，人 BRCA1 在 DT40 细胞中是功能性的，因为在人 BRCA1 敲入 DT40 细胞中，DNA 损伤诱导的 Rad51 焦点形成表明了这一点。这些结果表明，BRCA1 中心区域内的这些保守结构域对于 BRCA1 的 DNA 修复功能至关重要。这些发现为开发与 BRCA1 相关的乳腺癌新的治疗方法提供了有价值的工具。
