Abstract
DNA methyltransferases flip their target bases out of the DNA double helix for catalysis. Base flipping of C5-cytosine DNA methyltransferases was directly observed in the protein-DNA cocrystal structures of M.HhaI and M.HaeIII. Indirect structural evidence for base flipping of N6-adenine and N4-cytosine DNA methyltransferases was obtained by modeling DNA into the three-dimensional structures of M.TaqI and M.PvuII in complex with the cofactor. In addition, biochemical evidence of base flipping was reported for different N6-adenine DNA methyltransferases. As no protein-DNA cocrystal structure for the related N6-adenine and N4-cytosine DNA methyltransferases is available, we used light-induced photochemical cross-linking to identify the binding site of the extrahelical target bases. The N6-adenine DNA methyltransferases M.TaqI and M.CviBIII, which both methylate adenine within the double-stranded 5'-TCGA-3' DNA sequence, were photo-cross-linked to duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position in 50-60% and almost quantitative yield, respectively. Proteolytic fragmentation of the M. CviBIII-DNA complex followed by Edman degradation and electrospray ionization mass spectrometry indicates photo-cross-linking to tyrosine 122. In addition, the mutant methyltransferases M. TaqI/Y108A and M.TaqI/F196A were photo-cross-linked with 6-fold and 2-fold reduced efficiency, respectively, which suggests that tyrosine 108 is the primary site of modification in M.TaqI. Our results indicate a close proximity between the extrahelical target base and tyrosine 122 in M.CviBIII or tyrosine 108 in M.TaqI. As both residues belong to the conserved motif IV ((N/D/S)(P/I)P(Y/F/W)) found in all N6-adenine and N4-cytosine DNA as well as in N6-adenine RNA methyltransferases, a similar spatial relationship between the target bases and the aromatic amino acid residue within motif IV is expected for all these methyltransferases.
Highlights
S-adenosyl-L-methionine; DTT, dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; M.CviBIII, N6-adenine DNA methyltransferase from the Chlorella virus NC-1A; M.EcoRI, N6-adenine DNA methyltransferase from E. coli; M.HaeIII, C5-cytosine DNA methyltransferase from Haemophilus aegypticus; M.HhaI, C5-cytosine DNA methyltransferase from Haemophilus haemolyticus; M.PvuII, N4-cytosine DNA methyltransferase from Proteus vulgaris; M.TaqI, N6-adenine DNA methyltransferase from T. aquaticus; N-DNA Mtases, N6adenine and N4-cytosine DNA methyltransferases; N-DNA/RNA Mtases, N6-adenine and N4-cytosine DNA as well as N6-adenine RNA methyltransferases; ODN, oligodeoxyribonucleotide; PAGE, polyacrylamide gel electrophoresis
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]
Using a combination of Edman degradation and electrospray ionization mass spectrometry (ESI-MS) of a nucleopeptide obtained by proteolytic fragmentation of the covalent M.CviBIII-DNA complex we demonstrate that Tyr-122 is modified in the photo-cross-linking reaction
Summary
Methyltransferase; AdoMet, S-adenosyl-L-methionine; DTT, dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; M.CviBIII, N6-adenine DNA methyltransferase from the Chlorella virus NC-1A; M.EcoRI, N6-adenine DNA methyltransferase from E. coli; M.HaeIII, C5-cytosine DNA methyltransferase from Haemophilus aegypticus; M.HhaI, C5-cytosine DNA methyltransferase from Haemophilus haemolyticus; M.PvuII, N4-cytosine DNA methyltransferase from Proteus vulgaris; M.TaqI, N6-adenine DNA methyltransferase from T. aquaticus; N-DNA Mtases, N6adenine and N4-cytosine DNA methyltransferases; N-DNA/RNA Mtases, N6-adenine and N4-cytosine DNA as well as N6-adenine RNA methyltransferases; ODN, oligodeoxyribonucleotide; PAGE, polyacrylamide gel electrophoresis In this publication we show that the N6-adenine DNA Mtases M.TaqI from Thermus aquaticus [23, 24] and M.CviBIII from the Chlorella virus NC-1A [25], which both methylate adenine within the double-stranded 5Ј-TCGA-3Ј DNA sequence, give high photo-cross-linking yields with duplex ODNs containing 5-iodouracil at the target position (Scheme 1). Results from photo-cross-linking experiments with the mutant Mtases M.TaqI/
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