EcoRI DNA methyltransferase-DNA interactions

Abstract

We present a novel strategy with synthetic hemimethylated DNA substrates containing uracil for thymine and inosine for guanosine replacements and EcoRI DNA methyltransferase to characterize the importance of major groove hydrophobic groups to the sequence-specific modification of DNA. The bacterial Mtase uses S-adenosyl-L-methionine to methylate the double-stranded DNA site 5'GAATTC3' at the N6 position of the central adenosine of each strand. Uracil substitution in either strand at the outer thymine (5'GAATUC3') causes 2.2- and 1.7-fold improvements in specificity (kcat/KmDNA). The fact that the specificity constant for the substrate containing uracil in both strands is identical to the value expected for noninteracting substitutions suggests that no significant methyltransferase-DNA interactions are altered beyond the site of either substitution. Similar analysis of the internal thymine (5'GAAUTC3') also shows these methyl groups to make a negative contribution to specificity, although the observed nonadditivity with the doubly modified substrate clearly shows methyltransferase-DNA interactions beyond the site of substitution to be affected in this case. To further probe the effect of analogue incorporation on methyltransferase-DNA interactions beyond the site of substitution, the relatively "silent" and additive uracil changes (5'GAATUC3') were combined with inosine for guanosine substitutions (e.g., 5'IAATTC3') known to have significant negative effects on specificity. In contrast to the additivity observed with the outer thymines, these studies show significant changes in methyltransferase-DNA interactions caused by the removal of the thymine methyls. Our results implicate a complex and flexible methyltransferase-DNA interface in which subtle structural changes in the substrate are transmitted over the entire canonical site.(ABSTRACT TRUNCATED AT 250 WORDS)

Dam methylase fromEscherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates

“…For oligonucleotides these compensation temperatures Tc are higher: for the tetradecamers studied here Tc = 330.6 K (57°C) for the melting and cooling data. This value is even higher (Tc = 360 K) for a series of nonamers (52), but of the same order (Tc = 320 K) for another series of tetradecamers (11).…”

“…This was the case, even for dI substituting for dG, contrary to the observations of Martin et al (49) who had found that I-C base pairs were less stable than G.C and even than A.T pairs. In a recent study on hemimethylated oligonucleotides (11,50), however, the presence of I-C pairs instead of G.C pairs showed moderate to large increases in AH and AS, depending on the sequence and the (Fig. 4).…”

“…4). Such (11,52). Lumry and Rajender (53) have extensively reviewed previous work on small molecules and proteins and found that such 'enthalpy-entropy compensation was an ubiquitous property of water' and that the compensation temperature Tc obtained from the slope was always in the range between 250 and 315 K. They concluded that the compensation phenomena must be due to the properties of bulk water and differences in hydration states of the native and denatured form of a protein.…”

“…The interaction of EcoRI Mtase with hemimethylated DNA substrates containing base analogues has been investigated to characterise the contribution of individual functional groups toward DNA recognition by the enzyme (9)(10)(11). A similar approach has been applied in studies of M*EcoRl24I (13).…”

Simultaneous DNA Binding, Bending, and Base Flipping

“…The goal was to both extend the initial dye to dye distance so as to further remove any potential for direct protein-dye interactions, and to alter the orientation of the dye to the protein bound at the central hexanucleotide recognition segment. We previously demonstrated that M.EcoRI binds to two additional bases on each side of this recognition hexanucleotide (53), thus making contacts with 10 base pairs in the specific complex). Fig.…”