Structural analysis of the BisI family of modification dependent restriction endonucleases

Abstract The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups.

(C) The activity of full-length Eco15Iand Eco15I_Ntd were also compared.In (B) 30 ng of 550bp PCR product was incubated with 0.8-8pmol of Eco15I_Ntd or 0.9-9 pmol 12A for 40 minutes at 37 ºC.In (C) and 10 pmols of 20 bp long dsDNA were incubated with 5 pmols of protein for 1h at 37°C.Reaction conditions varied with salt concentration.The orientation of methyl groups is depicted schematicallyinternal 5mC is marked in pink and external 5mC is marked in green.The gels show representative data for at least 3 repeats.For the methyl group, a van der Waals radius of 2.0 Å was taken.

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Figure S1.Schematic representation of the proteins used in this study and design of the shortest active form of Eco15I (A, B) 6 or 12 N-terminal amino acids of Eco15I N-terminal domain were eliminated.The cleavage assay with recombinant proteins confirmed that only protein Eco15I_Ntd is active.Moreover, full-length NhoI was the subject of this study (A).(C)The activity of full-length Eco15Iand Eco15I_Ntd were also compared.In (B) 30 ng of 550bp PCR product was incubated with 0.8-8pmol of Eco15I_Ntd or 0.9-9 pmol 12A for 40 minutes at 37 ºC.In (C) and 10 pmols of 20 bp long dsDNA were incubated with 5 pmols of protein for 1h at 37°C.Reaction conditions varied with salt concentration.The orientation of methyl groups is depicted schematicallyinternal 5mC is marked in pink and external 5mC is marked in green.The gels show representative data for at least 3 repeats.

Figure S2 .
Figure S2.SPR response curves for the interaction of Eco15I_Ctd (A) and Eco15I_Ntd (B) with dsDNA oligonucleotide containing fully methylated target sequence.DNA was immobilized in the sensor at concentration of 2nM.Concentration of injected protein is marked in colors as indicated in the figure legend.Experiments were carried out in a buffer containing 50 mM KCl.

Figure S3 .
Figure S3.Eco15I_Ntd activity and binding to various substrates.Substrates contained a variable number of methyl groups and base substitutions within the enzyme recognition sequence.The orientation of methyl groups is depicted schematically.Internal and external 5mC bases are shown as pink and green balls, respectively.(A) Effect of the replacement of 5mC bases in the target sequence by T bases, paired with either A or G. (B) Comparison of substrates with two 5mC bases (and a substrate with four 5mC bases as a control).(C) Comparison of the activity of Eco15I_Ntd WT and mutants.In all cleavage assays (panels A, B, C) 10 pmols of DNA were incubated with 5 pmols of protein for 40 min at 37 °C, and digestion mixes were then analyzed by native polyacrylamide (PAA) electrophoresis.(D) Electrophoretic mobility shift assay (EMSA) to compare affinities of substrates with 5mC:G, T:G and T:A pairs. 1 pmol of DNA was incubated with increasing amounts (indicated below in pmol) of Eco15INtd for 30 min on ice.Theresults were visualized by autoradiography.All panels show representative data for at least 3 repeats.

Figure S4 .
Figure S4.NhoI activity for various concentrations of the enzyme.Oligonucleotides with the NhoI recognition sequence and 2-4 methyl groups were cleaved with increasing amounts of enzymes, and the reaction products were analyzed by native polyacrylamide (PAA) gels.The orientation of methyl groups is depicted schematicallyinternal 5mC is marked in pink and external 5mC is marked in green.10 pmols of DNA were incubated with 5 pmols of protein for 1h at 37°C.The gel shows representative data for 3 repeats.

Figure S5 .
Figure S5.Fitting of sensor response vs. concentration plot to the basic steady state affinity model, Biacore S200.The orientation of methyl groups is shown schematically.Experiments were carried out in a buffer containing either 50 mM or 150 mM KCl. Insets indicate the Kd in molar units, and "SE" stands for "standard error".

Figure S6 .
Figure S6.Multiple sequence alignment of BisI and its homologs.Residues responsible for divalent cations (Mg 2+ ) coordination are marked in blue, external methyl bindingin green and internal methyl bindingin pink.

Figure S7 .
Figure S7.Space filling representation of the binding pockets for external and internal methyl groups.Atoms are represented as balls with their van der Waals radii, and colored according to their atom type (carbons of the protein are green, and those of the 5mC are yellow).For the methyl group, a van der Waals radius of 2.0 Å was taken.

Figure S8 .
Figure S8.Substrate preference of NhoI mutant R84H.Digestions were performed with 200 ng of phage DNA and 150 ng of 3kb 5hmC-PCR product and 0.02-2 μg of enzyme at 37 ºC, for 1 h.The gel shows representative data for 3 repeats.

Figure S9 .
Figure S9.Solubility test of NhoI_V140A.The protein fractions obtained after lysis.T -total lysate, S -soluble fraction, P -insoluble fraction.The gel shows representative data for 3 repeats.

Figure S10 .
Figure S10.Comparison of the origin of BisI family enzymes and methyltransferases generating cognate target sequences.Methyltransferases with GCNGC, GCSGC or GCWGC specificity are marked in blue.BisI family members of activity confirmed in vitro are marked in orange and putative BisI family members are marked in red.Phylogeny was done using taxopy (https://github.com/apcamargo/taxopy)and the diagram was drawn with graphlan (https://github.com/biobakery/graphlan).

Table S1 .
Amino acid sequences of wild-type proteins used in the study.

Table S2 .
Primers used for site-directed mutagenesis of Eco15I_Ntd and NhoI.Codons for the amino acid substitutions are marked in bold.

Table S4 .
Data collection and refinement statistics.Statistics for the highest-resolution shell are shown in parentheses.