Improved Plasmid Vectors for the Analysis of Protein-Induced DNA Bending

Bending of DNA by proteins plays an important role in transcription initiation, DNA replication, and recombination. The degree of pro- tein-induced DNA bending is conveniently determined by combin- ing gel retardation techniques with the use of so-called bending vectors (1,2). Bending vectors contain duplicated circular permuted restric- tion sites and cloning sites for insertion of protein binding sequences. Restriction enzyme digestion readily generates fragments that are identical in size, but differ in the location of the binding site (Fig. 1). Open image in new window Fig. 1. Schematic representation of the pBend3-insert between the EcoRI-HindIII sites pBend3 was constructed by cloning of the 236 bp EcoRI-HindIII fragment of pBend2 (4) into pBluescript SK- (Stratagene). pBluescript is a high copy number plasmid and generates a large amount of DNA on plasmid extraction. The EcoRI- HindIII 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 in the upper part) is shifted. The sites XbaI and SalI (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.