DNA bending induced by Cro protein binding as demonstrated by gel electrophoresis

We report an approach for studying protein-induced DNA bends in solution that is based on measuring the sizes of circular DNA molecules by using two-dimensional gel electrophoresis. These circular fragments are obtained by ligating short synthetic oligonucleotides containing a protein-recognition region in the presence of protein. Oligonucleotides 21-base-pairs-long containing the O_R3 recognition site were synthesized and ligated in both the presence and the absence of the Cro repressor from λ phage. We show that in the presence of Cro protein, circular DNA molecules are formed with substantial frequency. No circular molecules are observed in the DNA samples ligated in the absence of Cro. These experiments clearly demonstrate that DNA bending is induced by Cro in this operator site. The sum of inherent plus Cro-induced bending is estimated as 45°.