Deuterium exchange of operator 8CH groups as a Raman probe of repressor recognition: interactions of wild-type and mutant lambda repressors with operator OL1.

The rate of deuterium exchange of a purine 8CH group in DNA is highly sensitive to both macromolecular secondary structure and intermolecular interactions which restrict solvent access to the major groove [Lamba, O.P., Becka, R., & Thomas, G.J., Jr. (1990) Biopolymers 29, 1465-1477]. We have exploited the sensitivity of the 8CH----8CD reaction to probe DNA recognition by the helix-turn-helix (HTH) motif of phage lambda cI repressor. We find that purine exchanges in the 19-base-pair OL1 operator are strongly and specifically restricted by binding of the HTH N-terminal domain of the repressor fragment (RF) comprising residues 1-102. The kinetics indicate large-scale obstruction of solvent access to operator 7N-8C purine sites. Interpretation of the exchange kinetics using a simple model suggests that only 7 purine residues (5 of 10 adenines and 2 of 9 guanines) remain unrestricted with respect to 8CH exchange in complexes of OL1 with the wild-type repressor. On the other hand, the 8CH exchange profile for the complex of OL1 with the Tyr88----Cys mutant repressor indicates that 9 purines (7 adenines and 2 guanines) are exchangeable. These results suggest important differences in major groove recognition in the two complexes. The proposed 8CH labeling profiles are consistent with molecular models of related complexes determined by X-ray crystallography [Jordan, S.R., & Pabo, C.O. (1988) Science 242, 893-899] and indicate that the structures observed in the crystal are largely maintained in solution.(ABSTRACT TRUNCATED AT 250 WORDS)