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

我在2001年发现BRCA 1与BARD 1构成RING异二聚体型泛素连接酶。在本研究中，我进一步研究了BRCA 1的底物，以阐明BRCA 1的活性如何有助于维持染色体稳定性的能力。我通过我们最初的筛选方法鉴定了几种底物。其中，我在2004年研究了核磷蛋白/B23（NPM）。BRCA 1-BARD 1在体内和体外与NPM相互作用并多聚泛素化。多聚泛素化是非传统的Lys-6连接型，因此NPM不会因修饰而降解，而是稳定化。BRCA 1-BARD 1和NPM在有丝分裂期间共定位于染色体表面，而NPM实际上在体内被泛素化。我认为NPM泛素化的缺乏可能是BRCA 1缺陷引起的中心体超扩增和非整倍体的基础。接下来，我在2005年分析了另一种候选底物RPB 8。BRCA 1-BARD 1在体内和体外与RPB 8相互作用并多聚泛素化。在稳定表达FLAG标记的RPB 8的HeLa细胞中，在UV照射后10分钟观察到RPB 8的泛素化，并且通过siRNA敲低BRCA 1来消除这种泛素化。有趣的是，稳定表达泛素抗性RPB 8突变体的HeLa细胞表现出UV超敏性。结果表明，BRCA 1通过RPB 8的泛素化，通过转录偶联的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.