Abstract
The dG residues within the EcoRI recognition sequence of ColE1 DNA have been selectively replaced with dI. Methylation of the altered sequence by the EcoRI modification enzyme is extremely slow as compared with methyl transfer to the natural recognition site. Since the affinity of the modification enzyme for the dI-containing sequence is considerably less than that for the natural sequence, we have concluded that the 2-amino group of dG has an important role in DNA site recognition by this enzyme. In contrast, the altered site is subject to cleavage by EcoRI endonuclease at rates essentially identical with those observed with the natural sequence. These results strongly suggest that the two enzymes utilize different contacts within the EcoRI site and are consisted with our conclusion (Rubin, R. A., and Modrich, P. (1977) J. Biol. Chem. 252, 7265-7272) that the two proteins interact with their common recognition sequence in different ways.
Highlights
The dG residues within the EcoRI recognition sequence of ColEl DNA have been selectively replaced with dI
In this paper we describe the highly selective substitution of deoxyinosine (d1) for the deoxyguanosine residues present in the EcoRI recognition sequence of ColEl DNA, a change which results in loss of the 2-amino group of guanine
Amino group of guanine for normal recognition within the EcoRI sequence is required by the methylase but not by the endonuclease. our conclusion
Summary
The dG residues within the EcoRI recognition sequence of ColEl DNA have been selectively replaced with dI. The altered site is subject to cleavage by EcoRI endonuclease at rates essentially identical with those observed with the natural sequence. These results strongly suggest that the two enzymes utilize different contacts within the EcoRI site and are consistent with our conclusion Since a variety of nucleotide analogs are available which form good WatsonCrick base pairs This method has been employed previously in studies with T7 RNA polymerase [5]. This work has demonstrated that this amino group is not required for sequence recognition and cleavage by the endonuclease, it does have an important role in recognition by the methylase
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