Monovalent cations regulate DNA sequence recognition by 434 repressor

The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's DNA-binding site modulate the affinity of DNA for protein. The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components. We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant O_R1 and O_R3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for O _R1, but not for O_R3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor-O_R1 complex requires the presence of monovalent cations; however, repressor-O_R3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect O_R1, but not O_R3, from·OH radical cleavage. Altering the relative length of the poly(dA)·poly(dT) tract in the non-contacted regions of the O_R1 and O_R3 can reverse the cation sensitivity of repressor's affinities for these two sites. Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins.