Mechanisms that regulate chromosomal stability through the ubiquitin lipase activity of BRCA1

我在2001年发现BRCA1与BARD1构成了RING异二聚体型泛素连接酶。在这项研究中，我进一步研究了 BRCA1 的底物，以阐明该活性如何促进 BRCA1 维持染色体稳定性的能力。我通过我们最初的筛选方法识别了几种基材。其中我在2004年研究了核磷蛋白/B23（NPM）。BRCA1-BARD1在体内和体外与NPM相互作用并被多泛素化。多泛素化是非传统的Lys-6连接类型，因此NPM不会因修饰而降解，而是稳定化。当 NPM 实际上在体内泛素化时，BRCA1-BARD1 和 NPM 在有丝分裂过程中共定位于染色体表面。我认为 NPM 泛素化的缺乏可能是 BRCA1 缺陷引起的中心体过度扩增和非整倍性的基础。接下来，我在 2005 年分析了另一种候选底物 RPB8。BRCA1-BARD1 在体内和体外与 RPB8 相互作用，并对 RPB8 进行多聚泛素化。在稳定表达 FLAG 标签的 RPB8 的 HeLa 细胞中，紫外线照射 10 分钟后观察到 RPB8 的泛素化，并且这种泛素化被 siRNA 敲低 BRCA1 所消除。有趣的是，稳定表达泛素抗性 RPB8 突变体的 HeLa 细胞表现出紫外线超敏性。结果表明，BRCA1 通过 RPB8 泛素化进行转录偶联 DNA 修复来调节染色体稳定性。

I have discovered in 2001 that BRCA1 constituted a RING heterodimer type ubiquitin ligase with BARD1. In this study I further investigated the substrates of BRCA1 to elacidate how the activity contributed the BRCA1's ability to maintain the chromosomal stability. I identified several substrates by our original screen methods. Among them I have investigated nucleophosmin/B23 (NPM) in 2004. BRCA1-BARD1 interacted with and polyubiquitinated NPM in vivo and in vitro. The polyubiquitination was non-traditional Lys-6-linked type, and therefore NPM was not degraded by the modification and instead stabilized. BRCA1-BARD1 and NPM were co-localized over the chromosomal surface during mitosis when NPM was actually ubiquitinated in vivo. I propose the lack of NPM ubiquitination could be the basis of centrosome hyper amplification and aneuploidy caused by BRCA1 deficiency. I next analyzed another candidate substrate, RPB8, in 2005. BRCA1-BARD1 interacted with and polyubiquitinated RPB8 in vivo and in vitro. The ubiquitination of RPB8 was observed 10 minutes after UV irradiation in HeLa cells stably expressing FLAG-tagged RPB8, and this ubiquitination was abolished by BRCA1 knockdown by siRNA. Interestingly HeLa cells stably expressing ubiquitin-resistant RPB8 mutant exhibited UV hypersensitivity. The results suggested that BRCA1 regulates chromosomal stability by transcription-coupled DNA repair through ubiquitination of RPB8.