Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI

Abstract

DNA methylation-dependent restriction enzymes have many applications in genetic engineering and in the analysis of the epigenetic state of eukaryotic genomes. Nevertheless, high-resolution structures have not yet been reported, and therefore mechanisms of DNA methylation-dependent cleavage are not understood. Here, we present a biochemical analysis and high-resolution DNA co-crystal structure of the N(6)-methyladenine (m6A)-dependent restriction enzyme R.DpnI. Our data show that R.DpnI consists of an N-terminal catalytic PD-(D/E)XK domain and a C-terminal winged helix (wH) domain. Surprisingly, both domains bind DNA in a sequence- and methylation-sensitive manner. The crystal contains R.DpnI with fully methylated target DNA bound to the wH domain, but distant from the catalytic domain. Independent readout of DNA sequence and methylation by the two domains might contribute to R.DpnI specificity or could help the monomeric enzyme to cut the second strand after introducing a nick.

Figure 1.

Biochemical validation of R.DpnI active site prediction. (A) Divalent metal ion dependence of the plasmid DNA cleavage by R.DpnI. The reaction buffer contained: no divalent metal ions, 10 mM MgCl₂, 10 mM CaCl₂ or 10 mM MnCl₂. (B) Cleavage activity of R.DpnI variants with single amino acid substitutions within the active site. M: molecular mass marker, control: non-cleaved substrate.

Figure 2.

Determination of R.DpnI sequence selectivity. (A) R.DpnI mediated cleavage of Dam and Hia5-methylated plasmid DNA in comparison to Dam-methylated and non-methylated DNA. R.DpnI concentration: 37.5 nM, pBR322 plasmid concentration: 5.6 nM, GATC sites concentration: 123.2 nM and SATS sites concentration: 537.6 nM. The wedge indicates decreasing concentration of R.DpnI (37.5 nM, 3.75 nM, 375 pM and 37.5 pM). M: molecular mass marker, c: non-cleaved DNA, Dam mDNA: Dam-methylated plasmid, nmDNA: non-methylated plasmid (isolated from a Dam⁻ E. coli strain), Hia5 mDNA: Hia5-methylated plasmid isolated from a Dam⁺ E. coli strain. (B) Sequence logo of R.DpnI selectivity on Dam (upper logo) and Dam and Hia5-methylated DNA (lower logo). pBR322 Dam or Dam⁺ Hia5-methylated plasmid was cleaved with R.DpnI and the resulting fragments were cloned to pUC18. A total of 61 independent clones were sequenced. Logos were generated by the WebLogo program (48).

Figure 3.

Binding of R.DpnI to different 21-mer oligo DNAs. Abbreviations for oligonucleotide duplexes: mDNA: fully methylated Gm6ATC, hmDNA: hemi-methylated Gm6ATC, nmDNA: non-methylated GATC, smDNA: methylated scrambled sequence (CTm6AG), ssmDNA: methylated sequence with scrambled positions 1 and 4 and unchanged m6A position (Cm6ATG). snmDNA: non-methylated scrambled sequence (CTAG). For full oligo sequences see Supplementary Table S4.

Figure 4.

Crystal structure of the R.DpnI-DNA complex. (A) Overview of the R.DpnI structure. The active site residues of the catalytic domain (blue) are indicated in magenta and the residues responsible for sequence recognition by the wH domain (green) are in yellow. Two loops that could not be reliably traced in the electron density (residues 46–49 and 131–136) were marked by the dotted lines. (B) Detail of the binding of the central m6A-T pairs to the winged helix domain. (C) Sequence recognition by the winged helix domain. The composite omit map was contoured at 1 σ.

Figure 5.

Binding assay of R.DpnI and its variants to different oligo DNAs. (A) Relative binding of different R.DpnI variants to methylated oligo DNA (mDNA). (B) Relative binding of the R.DpnI wH domain to different oligo DNAs. (C) Relative binding of the R.DpnI R231A variant to different oligo DNAs. The oligo coding is analogous as in Figure 3 (for data quantification see Supplementary Table S3, for sequences see Supplementary Table S4).

Figure 6.

Hemi-methylated DNA (cuthmDNA) cleavage by R.DpnI variants with alanine substitutions in the wH domain. Control: non-cleaved substrate. The sequence of the used oligo DNA is available in Supplementary Table S4. R.DpnI concentration: 37.5 nM. dsDNA concentration: 1 μM.

Figure 7.

Properties of the PD-(D/E)XK domain. (A) Cleavage of Dam-methylated and non-methylated pUC18 DNA by R.DpnI and its catalytic domain. (B) Cleavage of Dam and Dam and Hia5-methylated pBR322 DNA by R.DpnI and its catalytic domain. M: molecular mass marker, control: non-cleaved substrate, ‘+’: Dam-methylated DNA, ‘−’: non-methylated DNA, Dam: Dam-methylated plasmid, Hia5: Hia5-methylated plasmid isolated from a Dam⁺ E. coli strain. R.DpnI concentration: 37.5 nM, pUC18 plasmid concentration: 9.0 nM, pUC18 GATC sites concentration: 135 nM, pBR322 plasmid concentration: 5.6 nM, pBR322 GATC sites concentration: 123.2 nM and pBR322 SATS sites concentration: 537.6 nM.

Figure 8.

Pre-incubation of plasmid DNA with the R.DpnI wH domain. M: molecular mass marker, ‘−’: cleavage reaction without pre-incubation with the wH domain, ‘+’: cleavage reaction after pre-incubation with the wH domain (5-fold molar excess of wH domain over Gm6ATC sites was used).