Activity and structure of EcoKMcrA

Abstract

Escherichia coli McrA (EcoKMcrA) acts as a methylcytosine and hydroxymethylcytosine dependent restriction endonuclease. We present a biochemical characterization of EcoKMcrA that includes the first demonstration of its endonuclease activity, small angle X-ray scattering (SAXS) data, and a crystal structure of the enzyme in the absence of DNA. Our data indicate that EcoKMcrA dimerizes via the anticipated C-terminal HNH domains, which together form a single DNA binding site. The N-terminal domains are not homologous to SRA domains, do not interact with each other, and have separate DNA binding sites. Electrophoretic mobility shift assay (EMSA) and footprinting experiments suggest that the N-terminal domains can sense the presence and sequence context of modified cytosines. Pyrrolocytosine fluorescence data indicate no base flipping. In vitro, EcoKMcrA DNA endonuclease activity requires Mn2+ ions, is not strictly methyl dependent, and is not observed when active site variants of the enzyme are used. In cells, EcoKMcrA specifically restricts DNA that is modified in the correct sequence context. This activity is impaired by mutations of the nuclease active site, unless the enzyme is highly overexpressed.

Figure 1.

DNA cleavage by EcoKMcrA. The reactions were performed with 0.5 μM wild type EcoKMcrA (dimer) and 0.2 μM of unmodified, hemi- or fully methylated oligoduplex DNA or single stranded DNA, at 37°C in a buffer supplemented with 0.1 mM Mn²⁺. Top strand and bottom strand cleavage positions on the gels are marked by blue and red dotted lines, respectively. Gel lanes ‘S’ contained radiolabeled single-stranded oligonucleotides corresponding to the 5′-terminal fragments of the respective DNA strands (sizes in nucleotides are shown on the sides of the gels). The amounts of the bottom and top strand cleavage products after 3 hours of digestion are plotted as blue/red arrows along the oligoduplex sequences. The uncleaved oligonucleotides (the 0 hour lanes) contained a detectable fraction (∼10%) of shorter fragments. The reported cleavage data was corrected for the amount of these contaminants.

Figure 2.

Plasmid restriction assay. Quantification of the restriction of empty and M.HpaII carrying plasmids by E. coli cells expressing either wild type EcoKMcrA, two different active site variants of the enzyme, its N-terminal fragment lacking the catalytic domain or an unrelated protein (REM14). The pACYC184 plasmid carrying a chloramphenicol resistance gene was assayed in BL21(DE3) (McrA^-). The plasmid was either empty and thus without cytosine modifications or carried a gene for M.HpaII methyltransferase and thus was 5mC modified in the C5mCGG sequence context assumed to be among the EcoKMcrA targets.

Figure 3.

Structure of EcoKMcrA. (A) Ribbon diagram of the EcoKMcrA dimer in the asymmetric unit of the crystals, colored according to domains. The domain organization of each protomer of is shown below. (B) Comparison of a symmetrized model of the EcoKMcrA dimer based on the more elongated protomer, with small-angle X-ray scattering (SAXS) data for the protein in the absence of DNA. (C) EcoKMcrA model ab initio calculated from the SAXS data (grey spheres) overlaid with the symmetrized dimer that was used for the calculation of the predicted small angle X-ray scattering data presented in panel B.

Figure 4.

Structure of the N-terminal domain of EcoKMcrA. (A) Ribbon diagram in rainbow coloring from N- to C-terminus (blue to red). (B) Topology diagram generated with Pro-origami (50). (C) Electrostatic potential at the solvent excluded surface, blue represents positively charged regions, red negatively charged regions. (D) ConSurf (37) JTT conservation scores were mapped to the solvent accessible surface. Coloring is from cyan (poorly conserved regions) to magenta (for highly conserved regions). Panels A, C and D show the domain in the same orientation.

Figure 5.

DNA binding by the N-terminal fragment of EcoKMcrA (EcoKMcrA-N). (A) Electrophoretic mobility shift assay. DNA was unmodified, hemi- or fully methylated (central sequences shown on top; ‘M’ denotes 5mC). DNA concentration was 50 nM, concentrations of EcoKMcrA-N are indicated above gel lanes. Positions of free DNA and protein-DNA complexes are indicated. (B) DNase I footprint. Top: the region of the DNA protected from DNase I cleavage by EcoKMcrA-N binding is shown in bold font. The bottom strand G-35 position that becomes more susceptible to DNase I treatment upon EcoKMcrA-N binding is shown by ‘+’. Bottom: DNA protection by EcoKMcrA-N. Gel lanes ‘0’ contained untreated unmodified DNA, ‘1’ – the DNA treated with DNase I in the absence of EcoKMcrA-N, lanes ‘2’ and ‘3’ – the DNA treated with DNase I in the presence of 0.5 and 1 μM EcoKMcrA-N, respectively. Lanes ‘0m’, ‘1m’, ‘2m’ and ‘3m’ contained analogous samples prepared with hemimethylated DNA. Size markers (lanes ‘A+G’) were generated using a standard Maxam-Gilbert sequencing reaction. Positions of the DNA protected from DNase I cleavage upon EcoKMcrA-N binding are marked by blue (top strand) or red (bottom strand) dashes. The sequences of the protected regions are shown on the right-hand side of the gels.

Figure 6.

HNH domain dimer of EcoKMcrA. (A) Ribbon representation of the structure of the EcoKMcrA HNH domain. The structural Zn²⁺ ions, and the active site metal ions (Zn²⁺ in the crystal structure, and most likely Mn²⁺ in the physiological state), are shown together with their ligands. (B) Model of EcoKMcrA DNA complex, created based on the comparison with the Hpy99I restriction endonuclease. The blue and pink balls represent structural Zn²⁺ and active site metal ions, respectively. (C) Sequence alignment of the HNH core region of EcoKMcrA and related endonucleases: ScoMcrA (WP_011029780), TagI (WP_084609162), NmeCas9 (PDB: 5VGB), P1 phage Ref protein (PDB: 3PLW), human ZRANB3 endonuclease (PDB: 5MKW), T4 phage endonuclease VII (PDB: 1E7D) and PacI endonuclease (PDB: 3LDY). Zn – Zn²⁺ ligand, Me – active site metal ligand, Nu – nucleophile activator, Aux – auxiliary HNH residue.