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

我在2001年发现，BRCA1与BARD1共同构成了一种环状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，以确定该活性如何有助于BRCA1‘S维持染色体稳定的能力。我通过我们最初的筛选方法鉴定了几种底物。其中，我在2004年对核磷脂/B23(NPM)进行了研究。在体内和体外，BRCA1-BARD1与NPM相互作用，并使NPM多泛素化。多泛素化是非传统的Lys-6连接类型，因此NPM不会被修饰降解，而是稳定下来。在有丝分裂过程中，当NPM在体内泛素化时，BRCA1-BARD1和NPM共定位于染色体表面。我认为缺乏NPM泛素化可能是BRCA1缺乏导致中心体过度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基板RPB8。在体内和体外，BRCA1-BARD1与RPB8相互作用，并使RPB8多泛素化。紫外线照射10分钟后，在稳定表达FLAG标记的RPB8的HeLa细胞中观察到RPB8的泛素化，这种泛素化可被BRCA1被siRNA敲除。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反应。这些结果表明，BRCA1通过RPB8泛素化的转录偶联DNA修复来调节染色体的稳定性。

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.