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Structural basis of the methylation specificity of R.DpnI - PubMed

Structural basis of the methylation specificity of R.DpnI

Karolina Mierzejewska et al. Nucleic Acids Res. 2014 Jul.

Abstract

R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. 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. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. 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.

© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.

Overall comparison of the R.DpnI structures. The co-crystal structures of R.DpnI with either two DNA duplexes (A) or one DNA duplex (16) (B) per R.DpnI molecule with their catalytic domains oriented in the same way. PD-(D/E)XK and winged helix domains of the protein are shown in gold and green, respectively, and the DNA is in gray. The termini of the helices that are unwound in (A) but not in (B) are marked with black arrows..

Figure 2.
Figure 2.

Sequence recognition by the R.DpnI PD-(D/E)XK domain. Amino acids and DNA nucleotides are shown in all-atom representation. The composite omit map was contoured at 1.2σ. Panels (A–D) show the Gm6ATC bases of the proximal strand ordered as in the recognition sequence together with paired bases of the distal strand and interacting amino acids.

Figure 3.
Figure 3.

Methyl-methyl distance and DNA distortions in R.DpnI–DNA complex. The carbon-carbon distance between the methyl groups of m6A residues in the Gm6ATC sequence context was measured for idealized B-DNA (A), DNA in complex with the R.DpnI catalytic domain (B) and DNA in complex with the winged helix domain (C, D). Due to slightly better resolution, the density for the winged helix domain was clearer in the previous structure (16) (D), but the model is essentially identical to the current one (C). Both composite omit maps were contoured at 1.2σ.

Figure 4.
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.

Figure 5.
Figure 5.

Activity of R.DpnI variants. (A) R.DpnI and its variants were analyzed for protein purity by Coomassie-stained SDS-PAGE (1 μg per lane). (B) The catalytic activity of wild-type R.DpnI and its variants was tested against either non-methylated or Dam-methylated pBR322 plasmid as stated in the Materials and Methods section. M: molecular mass marker; C: non-cleaved substrate; WT: wild-type R.DpnI; ‘−’: non-methylated DNA; ‘+’: Dam-methylated DNA.

Figure 6.
Figure 6.

Change of deuteration upon addition of methylated DNA. Two molar equivalents of DNA and 10 s exchange time were used. Negative values indicate protection from hydrogen/deuterium exchange upon DNA binding and positive values indicate destabilization of hydrogen-bonding network of a given peptide. R.DpnI secondary structure is indicated (catalytic domain in yellow and winged helix domain in green). Active site residues are marked by yellow diamonds. Positions 229 and 231 that were mutated to weaken DNA binding are indicated by black triangles.

Figure 7.
Figure 7.

Methyl-methyl distances. Cumulative distribution for the carbon-carbon distance between methyl groups modeled in the transconformation on the two adenines of 114 structures in the PDB with the GATC sequence (and resolution ≤2.5 Å, R-factor ≤25%). For around 80% of all models, the distance between the methyl groups is below the 4.0 Å sum of methyl group van der Waals radii.

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