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 proteinʹs 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 OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor–OR1 complex requires the presence of monovalent cations; however, repressor–OR3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from radical dotOH radical cleavage. Altering the relative length of the poly(dA)·poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressorʹs 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.