Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States.
Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States.
Methods Enzymol. 2022;672:55-74. doi: 10.1016/bs.mie.2022.02.019. Epub 2022 Mar 25.
Formation of protein/nucleic acid complexes is essential for life. From DNA replication and repair to transcription and translation, myriad different proteins bind nucleic acids to execute their essential cellular functions. Our understanding of the mechanisms underlying recognition and processing of nucleic acids can be greatly informed by mapping protein domains and residues that form interfaces with their DNA or RNA targets. Here we describe a crosslinking protocol in which the unnatural amino acid p-benzoyl-l-phenylalanine (Bpa) integrated at selected sites within the PriA DNA helicase is used to map surfaces of the protein that interact with specific positions in a synthetic DNA replication fork in vitro.
蛋白质/核酸复合物的形成对生命至关重要。从 DNA 复制和修复到转录和翻译，无数不同的蛋白质与核酸结合以执行其基本的细胞功能。通过绘制与 DNA 或 RNA 靶标形成界面的蛋白质结构域和残基，我们可以更好地了解识别和处理核酸的机制。在这里，我们描述了一种交联方案，其中在 PriA DNA 解旋酶的选定位置整合非天然氨基酸 p-苯甲酰基-l-苯丙氨酸 (Bpa)，用于绘制该蛋白质与体外合成 DNA 复制叉中特定位置相互作用的表面。
