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Abstract:
The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent.
Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (
), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein- nucleic acid interactions observed for the EcoRI restriction endonuclease.
