Plasmid Vectors for the Analysis of Protein-Induced DNA Bending
互联网
396
Bending of DNA by proteins plays an important role in transcription initiation, DNA replication, and recombination. The degree of protein-induced DNA-bending can be simply and conveniently determined by combining gel electrophoresis of DNA-protein complexes with the use of special plasmid vectors carrying the bendable DNA sequence (1 ,2 ). The vectors contain duplicate sets of restriction sites in a direct repeat order and cloning sites for insertion of protein-binding sequences between the two sets. Restriction enzyme digestion readily generates DNA fragments, which are identical in size but differ in the location of the protein-binding site Fig. 1 ).
Fig. 1. Schematic representation of the pBend3 insert located between the EcoRI and Hin dIII sites. pBend3 was constructed by cloning of the 236-base-pair Eco RI-Hin dIII fragment of pBend2 (4 ) into pBluescript SK (Stratagene). pBluescript is a high-copy-number plasmid and generates a large amount of DNA upon plasmid extraction. The Eco RI-Hin dIII fragment contains 17 duplicated restriction sites. The duplicated sites can be used to generate DNA fragments of identical length, but in which the protein-binding sequence (gray rectangle) is shifted. The sites X baI and Sal I (in boxes) are unique and suitable for cloning of the protein binding sequence. Restriction sites are not drawn to scale. The sequence of the insert is shown in the lower part of the figure.
