Morris Joanna R, Solomon Ellen
Department of Medical and Molecular Genetics, Division of Genetics and Development, Guy's Kings and St Thomas' School of Medicine, King's College London, Guy's Hospital, London SE1 9RT, UK.
Hum Mol Genet. 2004 Apr 15;13(8):807-17. doi: 10.1093/hmg/ddh095. Epub 2004 Feb 19.
The N-terminus of the BRCA1 protein bears a RING finger domain that functions as an E3 ubiquitin ligase in vitro where it is able to catalyse the synthesis of monoubiquitin and polyubiquitin targeted proteins. This activity is greatly increased when BRCA1 is in a complex with its N-terminal binding partner BARD1. In this report we use an immunohistochemical approach to demonstrate the association of cellular BRCA1 with the end product of the ubiquitin conjugation and ligation pathway, conjugated ubiquitin. Association is apparent at DNA replication structures in S-phase and following treatment with hydroxyurea and also at sites of double strand break repair after exposure to ionizing radiation. Down-regulation of endogenous, cellular BRCA1 : BARD1 using siRNA results in abrogation of ubiquitin conjugation in these structures, suggesting that heterodimer activity is required for their formation. Conversely, ectopically expressed full-length BRCA1, but not BRCA1 bearing specific N-terminal amino acid substitutions, is able to cooperate with BARD1 to increase ubiquitin conjugation in cells. Conjugation of ubiquitin in foci is inhibited by the expression of ubiquitin bearing a lysine 6 mutation suggesting that the ubiquitin polymers formed at these sites are dependent on lysine-6 for linkage. Together these data demonstrate that BRCA1 directed ligation of ubiquitin occurs during S-phase and in response to replication stress and DNA damage and is therefore likely to be a significant aspect of BRCA1 cellular activity.
BRCA1蛋白的N端带有一个环状结构域，该结构域在体外作为E3泛素连接酶发挥作用，能够催化单泛素和多泛素靶向蛋白的合成。当BRCA1与其N端结合伴侣BARD1形成复合物时，这种活性会大大增强。在本报告中，我们使用免疫组织化学方法来证明细胞中的BRCA1与泛素缀合和连接途径的终产物——缀合泛素之间的关联。在S期的DNA复制结构处、用羟基脲处理后以及暴露于电离辐射后的双链断裂修复位点，这种关联都很明显。使用小干扰RNA（siRNA）下调内源性细胞BRCA1:BARD1会导致这些结构中的泛素缀合被消除，这表明异二聚体活性是它们形成所必需的。相反，异位表达的全长BRCA1，而不是带有特定N端氨基酸取代的BRCA1，能够与BARD1协同作用，增加细胞中的泛素缀合。带有赖氨酸6突变的泛素的表达会抑制病灶中的泛素缀合，这表明在这些位点形成的泛素聚合物的连接依赖于赖氨酸6。这些数据共同表明，BRCA1指导的泛素连接发生在S期以及对复制应激和DNA损伤的反应过程中，因此很可能是BRCA1细胞活性的一个重要方面。
