Perera Himasha M, Trakselis Michael A
Department of Chemistry and Biochemistry, Baylor University, Waco, Texas, 76798, USA.
Bio Protoc. 2020 Jun 20;10(12):e3649. doi: 10.21769/BioProtoc.3649.
The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay. Here, we describe a detailed protocol for site-specific DNA footprinting using 4-azidophenacyl bromide (APB). This method provides a straightforward, biochemical method to reveal the DNA binding orientation of MCM helicase and can be applied to other DNA binding proteins.
结合在DNA上的DNA结合蛋白的方向对于指导复合物中其他相关蛋白的组装以进行酶促作用具有决定性意义。例如，在复制体中，DNA解旋酶在复制叉处的方向指导其他相关复制体蛋白的组装。我们最近利用位点特异性DNA切割和作图分析确定了微型染色体维持（MCM）解旋酶在DNA叉处的方向。在这里，我们描述了一种使用4-叠氮基苯甲酰溴（APB）进行位点特异性DNA足迹分析的详细方案。该方法提供了一种直接的生化方法来揭示MCM解旋酶的DNA结合方向，并且可以应用于其他DNA结合蛋白。
