BRCA1-BARD1 及其变体通过核小体组蛋白泛素化来评估 E3 连接酶活性的方案。
Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation.
机构信息
Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
出版信息
STAR Protoc. 2024 Sep 20;5(3):103294. doi: 10.1016/j.xpro.2024.103294. Epub 2024 Sep 6.
The tumor suppressor breast cancer 1 (BRCA1) complexed with BRCA1-associated RING domain 1 (BARD1), a RING-type E3 ligase, facilitates the attachment of ubiquitin onto the substrate protein. Here, we present a protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation. We describe steps for isolating 147 bp Widom 601 DNA and assembling nucleosome core particles (NCPs). We then detail procedures for the in vitro ubiquitylation of nucleosome histone H2A by BRCA1-BARD1 and its variants. For complete details on the use and execution of this protocol, please refer to Wang et al..
肿瘤抑制因子乳腺癌 1（BRCA1）与 BRCA1 相关的环指结构域 1（BARD1）形成复合物，BARD1 是一种 RING 型 E3 连接酶，可促进泛素连接到底物蛋白上。在这里，我们提供了一种通过核小体组蛋白泛素化来评估 BRCA1-BARD1 及其变体的 E3 连接酶活性的方案。我们描述了分离 147bp Widom 601 DNA 和组装核小体核心颗粒（NCP）的步骤。然后，我们详细介绍了 BRCA1-BARD1 及其变体体外对核小体组蛋白 H2A 进行泛素化的程序。有关此方案使用和执行的完整详细信息，请参见 Wang 等人的研究。
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