Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC)

Abstract

A set of dA and T analogue suitable for the study of protein DNA interactions have been incorporated into the central d(ATAT) sequence within d(GACGATATCGTC). The individual analogues have one potential protein contact (either a hydrogen-bonding group of a CH3 group capable of a van der Waals interaction) deleted. In general, the modified bases do not perturb the overall structure of the dodecamer, enabling results obtained to be simply interpreted in terms of loss of protein DNA contacts. We have used the modified oligodeoxynucleotide set to study the recognition of DNA by the EcoRV restriction endonuclease [recognition sequence d(GATATC)]. The kcat and Km values for the set have been determined, and a comparison with results seen with the parent oligodeoxynucleotide (containing no modified bases) has been carried out. Three classes of results are seen. First, some analogues lead to no change in kinetic parameters, meaning no enzyme contact at the altered site. Second (this is seen for most of the modified oligodeoxynucleotides), a drop in the kcat/Km ratio relative to the parent is observed. This comes mainly from a decrease in kcat, implying that the endonuclease uses the interaction under study to lower the transition-state barrier rather than to bind the substrate. Analyses of these results show that the drop in kcat/Km is what would be expected for the simple loss of hydrogen bond or a CH3 contact between the enzyme and the oligodeoxynucleotide. This implies a contact of these types at these sites. Third, some analgoue-containing oligodeoxynucleotdes are not substrates; i.e., the kcat/Km drop is much greater than would be expected for loss of a single hydrogen bone or CH3 contact. These results are interpreted in terms of a cooperative mechanism whereby the loss of one interaction causes a rearrangement at the enzyme active site leading to a consequent loss of further protein substrate contacts. However, in these cases gross structural changes in the oligodeoxynucleotide conformation cannot be excluded. It is found that the endonuclease makes very many interactions to the d(ATAT) sequence within its d(GATATC) recognition site, and these occur in both the major and minor grooves. The results obtained have been used to explain how the enzyme achieves the high degree of cleavage specificity for d(GATATC) as compared to all other sequences.