DNA Distortion and Base Flipping by the EcoRV DNA Methyltransferase

Abstract

The EcoRV DNA methyltransferase introduces a CH 3 group at the 6-amino position of the first dA in the duplex sequence d(GATATC). It has previously been reported that the methylase contacts the four phosphates (pNpNpGpA) at, and preceding, the 5-end of the recognition sequence as well as the single dG in this sequence (Szczelkun, M. D., Jones, H., and Connolly, B. A. (1995) Biochemistry 34, 10734 -10743). To study the possible role of the dA and T bases within the ATAT sequence, interference studies have been carried out using diethylpyrocarbonate and osmium tetroxide. The methylase bound very strongly to hemimethylated oligonucleotides modified at the second AT, of the ATAT sequence, in the unmethylated strand of the duplex. This probably arises because these modifications facilitate DNA distortion that follows the binding of the nucleic acid to the protein. Oligonucleotides containing modified bases at both the target dA base and its complementary T were used to determine whether this dA methylase flips out its target base in a similar manner to that observed for dC DNA methylases. In binary EcoRV methylase-DNA complexes, analogues that weakened the base pair caused an increase in affinity between the protein and the nucleic acid. In contrast, in ternary EcoRV methylase-DNA-sinefungin (an analogue of the natural co-factor, S-adenosyl-L-methionine (AdoMet)) complexes, only small differences in affinity were observed between the normal dA-T base pair and the analogues. These results are almost identical to those seen with DNA dC methylases (Klimasauskas, S., and Roberts R.

Functional Roles of Conserved Amino Acid Residues in DNA Methyltransferases Investigated by Site-directed Mutagenesis of theEcoRV Adenine-N6-methyltransferase

“…Five of these variants do not bind to DNA, four of which carry a mutation in the putative DNA binding domain (42). Catalytic Mechanism-There is evidence that N-MTases flip out their target adenine prior to methyl group transfer (31)(32)(33)(34). Thus, the active site of the enzyme must form a binding site for the flipped out adenine residue, providing a hydrophobic pocket and hydrogen bond partners for the adenine as well as catalytic residues.…”

“…Interestingly, the conserved DPPY motif structurally corresponds to motif IV in C-MTases, which contains the catalytic cysteine residue. It is likely that N-MTases, like C-MTases, flip their target base out of the DNA double helix (31)(32)(33)(34).…”

The Mechanism of DNA Cytosine-5 Methylation

“…It turns out that there is no common rule, and particular classes or even individual enzymes appear to have different solutions to the problem. Kinetic studies of the adenine-N-6 MTases Ecodam (40), EcoRI (41), EcaI (42), and EcoRV (43) suggest that these enzymes show little, if any, orientation preference during catalysis, although some binding preference for hemimethylated versus unmethylated sites has been reported for M.EcoRV (44) and M.RsrI (45). Leaving aside the eukaryotic C5-MTases for which selectivity for hemimethylated DNA is crucial for their maintenance function (46,47), the bacterial C5-MTases also show clear differences in how unmethylated and hemimethylated substrates are processed (48,49).…”

Identification of the Binding Site for the Extrahelical Target Base in N 6-Adenine DNA Methyltransferases by Photo-cross-linking with Duplex Oligodeoxyribonucleotides Containing 5-Iodouracil at the Target Position

“…Biochemical evidence for a base flipping mechanism of the N 6 -adenine DNA Mtases M.EcoRI (11) and M.TaqI (12) was obtained using duplex oligodeoxyribonucleotides (ODNs) containing the fluorescent base analogue 2-aminopurine at the target positions. In addition, tighter binding of the N 6 -adenine DNA Mtases M.EcoRV (13) and M.EcoRI (14) to duplex ODNs carrying base analogues with reduced WatsonCrick hydrogen bonding potential at the target positions was observed and attributed to a reduced energetic cost to flip out the target base. Furthermore, a thymine residue placed at the target position within a duplex ODN showed an enhanced reactivity toward potassium permanganate oxidation in the presence of M.TaqI, which was interpreted by a higher accessibility of the thymine residue in the binary complex due to base flipping (15) To identify the binding site of the extrahelical target base in N 6 -adenine DNA Mtases, we used ultraviolet light-induced photochemical cross-linking, which is a powerful technique to define specific contact points in nucleoprotein complexes, where direct structural information is not available (for a review, see Ref.…”