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Structural basis of the methylation specificity of R.DpnI.

Mierzejewska K, Siwek W, Czapinska H, Kaus-Drobek M, Radlinska M, Skowronek K, Bujnicki JM, Dadlez M, Bochtler M - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.

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Related in: MedlinePlus

Recognition of the 6-methyladenines by R.DpnI. Interaction of the m6A bases with the R.DpnI catalytic (A, B) and winged helix (C, D) domains. The methyl groups are in contact with a long loop connecting adjacent antiparallel β-strands in the case of the catalytic domain and an α-helix in the case of the winged helix domain. For clarity, a part of protein was omitted in panel (B) and parts of DNA in all panels.
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Figure 4: Recognition of the 6-methyladenines by R.DpnI. Interaction of the m6A bases with the R.DpnI catalytic (A, B) and winged helix (C, D) domains. The methyl groups are in contact with a long loop connecting adjacent antiparallel β-strands in the case of the catalytic domain and an α-helix in the case of the winged helix domain. For clarity, a part of protein was omitted in panel (B) and parts of DNA in all panels.

Mentions: There is only a single hydrogen bond between the R.DpnI catalytic domain and the two central base pairs of its target sequence, which is donated by the Gln18 Nϵ atom to the O2 atom of the distal thymine (Figure 2B and Supplementary Figure S4). As G:C pairs would have an amino group in the central minor groove position clashing with the Gln18 Nϵ (which is in turn held in place by other hydrogen-bonding interactions), this contact likely contributes to sequence selectivity. All other polar interactions with the bases of the m6A:T pairs are solvent mediated (Figure 2B and C). The main van der Waals contacts between R.DpnI and the inner m6A:T pairs are made by the m6A methyl groups. As the methyl groups are very close to each other, they are bound together in one cleft of R.DpnI catalytic domain (Figure 4 and Supplementary Figure S5). The hydrophobic pocket is formed by Leu129, Arg135 and Trp138, which are all located in the long loop that gets ordered upon DNA binding. Trp138 plays a key role in the recognition of m6A methylation. It simultaneously interacts with both methyl groups: its pyrrole ring is in van der Waals contact with the m6A of the proximal DNA strand and its benzene ring with the m6A of the distal strand. The conformation of Trp138 side chain is stabilized by packing against Gly140 (particularly the NH group, glycine in this position is required because the Cβ atom of any other amino acid would clash with the indole ring of Trp138) and by a solvent-mediated interaction of its Nϵ atom with the m6A phosphate (Figures 2 and 4 and Supplementary Figure S4).


Structural basis of the methylation specificity of R.DpnI.

Mierzejewska K, Siwek W, Czapinska H, Kaus-Drobek M, Radlinska M, Skowronek K, Bujnicki JM, Dadlez M, Bochtler M - Nucleic Acids Res. (2014)

Recognition of the 6-methyladenines by R.DpnI. Interaction of the m6A bases with the R.DpnI catalytic (A, B) and winged helix (C, D) domains. The methyl groups are in contact with a long loop connecting adjacent antiparallel β-strands in the case of the catalytic domain and an α-helix in the case of the winged helix domain. For clarity, a part of protein was omitted in panel (B) and parts of DNA in all panels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117772&req=5

Figure 4: Recognition of the 6-methyladenines by R.DpnI. Interaction of the m6A bases with the R.DpnI catalytic (A, B) and winged helix (C, D) domains. The methyl groups are in contact with a long loop connecting adjacent antiparallel β-strands in the case of the catalytic domain and an α-helix in the case of the winged helix domain. For clarity, a part of protein was omitted in panel (B) and parts of DNA in all panels.
Mentions: There is only a single hydrogen bond between the R.DpnI catalytic domain and the two central base pairs of its target sequence, which is donated by the Gln18 Nϵ atom to the O2 atom of the distal thymine (Figure 2B and Supplementary Figure S4). As G:C pairs would have an amino group in the central minor groove position clashing with the Gln18 Nϵ (which is in turn held in place by other hydrogen-bonding interactions), this contact likely contributes to sequence selectivity. All other polar interactions with the bases of the m6A:T pairs are solvent mediated (Figure 2B and C). The main van der Waals contacts between R.DpnI and the inner m6A:T pairs are made by the m6A methyl groups. As the methyl groups are very close to each other, they are bound together in one cleft of R.DpnI catalytic domain (Figure 4 and Supplementary Figure S5). The hydrophobic pocket is formed by Leu129, Arg135 and Trp138, which are all located in the long loop that gets ordered upon DNA binding. Trp138 plays a key role in the recognition of m6A methylation. It simultaneously interacts with both methyl groups: its pyrrole ring is in van der Waals contact with the m6A of the proximal DNA strand and its benzene ring with the m6A of the distal strand. The conformation of Trp138 side chain is stabilized by packing against Gly140 (particularly the NH group, glycine in this position is required because the Cβ atom of any other amino acid would clash with the indole ring of Trp138) and by a solvent-mediated interaction of its Nϵ atom with the m6A phosphate (Figures 2 and 4 and Supplementary Figure S4).

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.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.

Show MeSH
Related in: MedlinePlus