DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.

DNA methyltransferases (Mtases) 1 are a biologically important class of enzymes that catalyze the transfer of the activated methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the N-6 nitrogen of adenine and the N-4 nitrogen of cytosine or the C-5 carbon of cytosine within their DNA recognition sequences (1). As DNA methylation is a postreplicative process that depends on the presence or regulation of DNA Mtases, a particular DNA sequence may exist in its fully methylated, its unmethylated, or transiently in its hemimethylated form. Thus, DNA methylation can be regarded as an increase of the information content of DNA (2), which serves a wide variety of biological functions, including protection of the host genome from endogenous restriction endonucleases, DNA mismatch repair after replication, regulation of gene expression and DNA replication, embryonic development, genomic imprinting, and X-chromosome inactivation (3-5). Furthermore, it plays a role in carcinogenesis (6). Three-dimensional structures are available for the C 5 -cytosine DNA Mtases M.HhaI (7) and M.HaeIII (8) in complex with DNA. Both enzymes consist of two domains forming a positively charged cleft, which accommodates the DNA. However, the most striking feature of these protein-DNA complexes is that the target cytosines are completely rotated out of the DNA double helix and placed in a pocket within the cofactor binding domains, where catalysis takes place. In addition, the structures of the N 6 -adenine DNA Mtases M.TaqI (9) and the N 4 -cytosine DNA Mtase M.PvuII (10) in the absence of DNA were reported. These N 6 -adenine and N 4 -cytosine DNA Mtases (N-DNA Mtases) also have a bilobal structure forming a positively charged cleft. Modeling B-DNA in this cleft showed that the distance between the target base and the bound AdoMet is too large for a direct methyl group transfer, and a base flipping mechanism as observed for the C 5 -cytosine DNA Mtases was postulated. 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 * This work was supported by a grant from the Deutsche Forschungsgemeinschaft (We1453/3-2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

The kinetics of the interaction of Rab7 with REP-1 have been investigated using the fluorescence of GDP and GTP analogs at the active site of Rab7. The results show that REP-1 has higher affinity for the GDP bound form of Rab7 (K d = 1 nM) than for the GTP bound form (K d = 20 nM). Both affinities should still be sufficient for the formation of stable complexes in the cell. The association reaction proceeds in two steps for the GDP bound form. The initial step is fast (k +1 = ca. 10 U M 3I s 3I ) and concentration dependent while the second represents a slow equilibration (k +2 +k 3 32 = 3.5 s 31 ) which has little effect on the overall equilibrium. The difference in affinity of the two nucleotide bound forms arises from a difference in dissociation rates (0.012 s 3I for Rab7cGDP and 0.2 s 3I for Rab7cGTP). z 1998 Federation of European Biochemical Societies.