Structural basis of the methylation specificity of R.DpnI.
Bottom Line: We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict.The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains.We also present hydrogen/deuterium exchange data that support 'crosstalk' between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity.
Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.Show MeSH
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Mentions: The G:C pairs of the Gm6ATC target sequence hydrogen bond extensively with R.DpnI (Figure 2 and Supplementary Figure S4). The proximal guanine is hydrogen bonded via its O6 and N7 atoms with the Nϵ and Nζ atoms of Arg135 (which also forms part of the hydrophobic pocket for the methyl groups) and via its N2 atom with the Oδ atom of Asn48 (Figure 2A). The distal guanine accepts two hydrogen bonds to its O6 and N7 atoms from the Arg126 guanidino group. The complementary cytosine donates a hydrogen bond from its N4 to the main chain carbonyl of Asp78 of the first β-strand in the β-sheet. On the minor groove side this C:G base pair is recognized by a single hydrogen bond from the Ser17 Oγ of the N-terminal α-helix to the cytosine O2 atom (Figure 2D). We also observe two additional hydrogen bonds between flanking cytosines on both sides of the target sequence and Asn48 and Asn77, which according to the biochemical data do not contribute to sequence specificity (16,28).
Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.