DNA 亲和纯化：一种用于鉴定和分析与核酸结合的蛋白质的下拉分析方法。

DNA Affinity Purification: A Pulldown Assay for Identifying and Analyzing Proteins Binding to Nucleic Acids.

机构信息

Molecular Oncology, Faculty of Medicine, Leipzig University, Leipzig, Germany.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA, USA.

出版信息

Methods Mol Biol. 2021;2267:81-90. doi: 10.1007/978-1-0716-1217-0_6.

DOI:10.1007/978-1-0716-1217-0_6

PMID:33786786

Abstract

The interaction of proteins with DNA plays a central role in gene regulation. We describe a DNA affinity purification method that allows for identification and analysis of protein complex components. For example, a DNA probe carrying a transcription factor binding site is used to purify proteins from a nuclear extract. The proteins binding to the probe are then identified by mass spectrometry. In similar experiments, proteins purified by this pulldown method can be analyzed by Western blot. Employing this method, we found that the DREAM transcriptional repressor complex binds to CHR transcriptional elements in promoters of cell cycle genes. This complex is important for cell cycle-dependent repression and as part of the p53-DREAM pathway serves as a link for indirect transcriptional repression of target genes by the tumor suppressor p53. In general, the methods described can be applied for the identification and analysis of proteins binding to DNA.

摘要

蛋白质与 DNA 的相互作用在基因调控中起着核心作用。我们描述了一种 DNA 亲和纯化方法，可用于鉴定和分析蛋白质复合物的组成部分。例如，携带转录因子结合位点的 DNA 探针可用于从核提取物中纯化蛋白质。然后通过质谱法鉴定与探针结合的蛋白质。在类似的实验中，通过这种下拉法纯化的蛋白质可以通过 Western blot 进行分析。使用这种方法，我们发现 DREAM 转录抑制复合物结合到细胞周期基因启动子中的 CHR 转录元件上。该复合物对于细胞周期依赖性抑制很重要，并且作为 p53-DREAM 途径的一部分，它是肿瘤抑制因子 p53 对靶基因进行间接转录抑制的连接。一般来说，所描述的方法可用于鉴定和分析与 DNA 结合的蛋白质。