Leng Fenfei
Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.
Methods Mol Biol. 2013;1054:267-82. doi: 10.1007/978-1-62703-565-1_18.
Protein-induced DNA bending plays a key role in many essential biological processes, such as DNA replication, recombination, and transcription, and can be analyzed by a variety of biochemical and biophysical methods, such as electrophoresis mobility shift assay (EMSA), X-ray crystallography, nuclear magnetic resonance (NMR), and DNA ring closure assay. In this chapter, I will provide a detailed protocol for studying protein-induced DNA bending by utilizing the plasmid pBendAT. pBendAT carries a 230 bp DNA segment containing five pairs of restriction-endonuclease recognition sites and can be used to produce a set of five DNA fragments of identical length, with each fragment having a different positioning of a protein-binding site. Binding of a protein to this site will divide a DNA fragment into two DNA segments. If protein binding leads to DNA bending, then the two DNA segments will be at an angle to each other and the distance between the DNA ends will shorten. As a result, the gel mobility of the protein-DNA complex will be affected as the mobility of a rigid DNA fragment is inversely proportional to the end-to-end distance. Therefore, by analyzing how the position of protein binding within a fragment affects the gel mobility of the protein-DNA complexes, we are able to determine the DNA bending angle and the location of the bend. The DNA fragments of identical length can also be conveniently generated by PCR amplification using pBendAT as the DNA template. Since the 230 bp DNA fragment of pBendAT does not contain more than two consecutive AT base pairs, pBendAT is particularly suitable for the assessment of DNA bending induced by proteins recognizing AT-rich DNA sequences cloned in the 230 bp DNA fragment.
蛋白质诱导的DNA弯曲在许多重要的生物学过程中起着关键作用，如DNA复制、重组和转录，并且可以通过多种生化和生物物理方法进行分析，如电泳迁移率变动分析（EMSA）、X射线晶体学、核磁共振（NMR）以及DNA环化分析。在本章中，我将提供一个利用质粒pBendAT研究蛋白质诱导的DNA弯曲的详细方案。pBendAT携带一个230 bp的DNA片段，其中包含五对限制性内切酶识别位点，可用于产生一组五个长度相同的DNA片段，每个片段具有不同的蛋白质结合位点定位。蛋白质与该位点的结合会将一个DNA片段分成两个DNA片段。如果蛋白质结合导致DNA弯曲，那么这两个DNA片段将相互成一定角度，并且DNA末端之间的距离会缩短。结果，蛋白质-DNA复合物的凝胶迁移率会受到影响，因为刚性DNA片段的迁移率与端到端距离成反比。因此，通过分析片段内蛋白质结合位置如何影响蛋白质-DNA复合物的凝胶迁移率，我们能够确定DNA弯曲角度和弯曲位置。使用pBendAT作为DNA模板，通过PCR扩增也可以方便地生成长度相同的DNA片段。由于pBendAT的230 bp DNA片段不包含超过两个连续的AT碱基对，pBendAT特别适合评估由识别克隆在230 bp DNA片段中的富含AT的DNA序列的蛋白质诱导的DNA弯曲。
