Role of the Z-Amino Group of Deoxyguanosine in Sequence Recognition by EcoRI Restriction and Modification Enzymes*

The dG residues within the EcoRI recognition sequence of ColEl 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 Z-amino group of dG has an important role in DNA site recognition by this enzyme. In con-trast, 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 (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.

The dG residues within the EcoRI recognition sequence of ColEl 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 Z-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 consistent 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.
As summarized in the previous paper (I), EcoRI endonuclease and methylase are proteins of quite distinct physical properties which interact with a common, 2-fold symmetric hexanucleotide sequence in duplex DNA (2,3). However, the molecular mechanisms involved in specific sequence recognition by these proteins are not known. One approach to this problem is to study the effects of nucleotide analog substitution within the recognition sequence. Since a variety of nucleotide analogs are available which form good Watson-Crick base pairs (41, their utilization may permit identification of DNA functional groups involved in sequence recognition. This method has been employed previously in studies with T7 RNA polymerase (5). In this case, however, analog substitutions were extensive and thus may have resulted in significant alteration of general polynucleotide structure. In this paper we describe the highly selective substitution of deoxyinosine (d1) for the deoxyguanosine (dG) residues present in the EcoRI recognition sequence of ColEl DNA, a change which results in loss of the 2-amino group of guanine * This investigation was supported by United States Public Health Service Grant GM23719 and Grant PCM76-04914 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "'uduertisement" in accordance with 18 U. S. C. Section 1734 solely to indicate this fact.

RESULTS
Sequence about ColEl EcoRI Site-The 3'-nucleotide at termini generated by EcoRI endonuclease cleavage is dG (2). Therefore, these termini can be specifically labeled by incubation with [&'P]dGTP and T4 DNA polymerase, which catalyzes an exchange reaction at 3'-termini of duplex DNA (8,12). As shown in Table I  in 3'-dAMP and 3'-dGMP, with label in the adenine nucleotide being i.6 to 1.7 times that found in 3'-dGMP. Recovery of label in dGMP demonstrates that dG is in fact present as a penultimate nucleotide. Furthermore, the recovery of approximately twice as much 32P in 3'-dAMP as in 3'-dGMP indicates that dA is present on the 5' side of dG at both ends of the linear molecule. The only sequence for ColEl EcoRI site consistent with these results is where sites of cleavage and methylation by EcoRI enzymes are indicated by arrows and asterisks, respectively (2, 3).
Construction and Characterization of dZ-substituted Molecules -The method employed for selective substitution of d1 within the EcoRI site of ColEl DNA is summarized in Fig. 1. Control molecules were prepared in an identical fashion except [ &*P]dGTP replaced [ a-32P]dITP in the T4 polymerasecatalyzed exchange reaction.
Since the penultimate dG on the 5'-side of one endonuclease cleavage point lies outside the minimal EcoRI recognition sequence, we wished to minimize exchange into this position. However, it was also necessary to ensure that essentially all termini participated in the exchange reaction. As shown in Table II (Table  III). However, The presence of d1 as the 3'-terminal nucleotide of the EcoRI-cleaved substrate analog was confirmed using T4 DNA polymerase. If ["'P]dIMP is present within the EcoRI site of the analog, then subsequent to cleavage with EcoRI endonuclease label should be released as 5'-dIMP upon incubation with the polymerase and dGTP via the terminal exchange reaction. Moreover, this release of label from DNA should not occur in the presence of dATP since this nucleotide would allow the polymerase to extend EcoRI-generated 3'-termini (Fig. 1) in a reaction that is much faster than exchange (8). As shown in Table IV, release of nnP occurred only after prior treatment of dI-containing circles with the endonuclease and was suppressed by the presence of dATP but in the presence of dGTP was complete, yielding dIMP exclusively. Although not shown, analogous experiments with dG-substituted DNA yielded identical results except that azP was recovered only in 5'-dGMP.
Thus within a recognition sequence containing d1. Furthermore, since the endonuclease reactions described here were performed at subsaturating DNA concentrations (15% of the K,,, concentration determined previously for ColEl DNA (6)), the kinetic parameters for cleavage within a dI-containing sequence cannot be less favorable than those for cleavage within the natural site. Therefore, the 2-amino group of deoxyguanosine does not have an important role in recognition and catalysis by this enzyme.

Methylase
Activity at EcoRI Sites Containing dl-In contrast to results obtained with the endonuclease, the presence of d1 within the EcoRI recognition sequence resulted in a marked reduction in the rate of methylation (Fig. 3). Under steady state conditions, the rate of methylation of dI-containing DNA circles was only 4% of that observed with dGsubstituted control circles or with natural ColEl DNA. To determine whether this low level of activity reflected methylation of sequences containing d1 as opposed to methylation of a small fraction of residual sites with dG, methyl transfer was examined as a function of enzyme concentration (Fig. 4).