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Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix - PubMed

  • ️Wed Jan 01 2014

. 2014 Oct 29;42(19):12092-101.

doi: 10.1093/nar/gku871. Epub 2014 Sep 27.

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Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

John R Horton et al. Nucleic Acids Res. 2014.

Abstract

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.

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

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Figures

Figure 1.
Figure 1.

Packing interaction of the MspJI–DNA complex in the crystal lattice. (a) Three tetramer–DNA complexes packed together in space group P61. Each complex is rotated 120° along the crystallographic c-axis. (b) One tetramer interacts with two symmetry-related DNA molecules. (c) Only the C-D dimer of the tetramer interacts with DNA. (d) Alternatively, one DNA molecule interacts with two symmetry-related tetramers. (e) The 27-bp DNA oligo used for crystallization. (f) The subunits C and D of two different tetramers interact with one DNA molecule.

Figure 2.
Figure 2.

General features of MspJI–DNA interaction. (a) One tetramer interacts with two DNA molecules. The A-B dimer is in closed conformation (bottom), while the C-D dimer is in the open conformation (top). The DNA bound specifically by the recognition domain of subunit C (in blue on the left side of the tetramer) is aligned with, and closest to, the catalytic center formed by subunits A (green) and D (see panel (b)). (b) Simplified tetramer formation mediated by the four catalytic domains (after removing all four DNA recognition domains). The DNA molecule on the left side of the tetramer is close to the catalytic center formed by subunits A and D (dashed red circle). In our structure, the DNA is moved away, due to its interactions with the adjacent tetramer. (c, d) Two views of the recognition domain of subunit C binding DNA specifically. The side chains of Gln33 and Glu65 are located in the minor DNA groove, while Lys173 is in the next major groove.

Figure 3.
Figure 3.

Details of MspJI–DNA interaction. (a) The recognition domain of subunit C binds specifically to 5mCNNG. The flipped 5mC is in an acidic slot-like pocket. The surface charge distribution at neutral pH is displayed as blue for positive, red for negative and white for neutral. (b) Schematic MspJI–DNA interactions via subunit C (blue) close to the 5′ end of the modified strand (at base-pair position 5) and subunit D of second tetramer (green) at base-pair position 18. (c, d) Two views of specific interactions involving the flipped 5mC in the slot-like pocket. Inter-atom distances are shown in Å. (e) DNA base interactions involving Gln33, Glu65 and Lys173. (f) The recognition domain of subunit D binds non-specifically GNNG sequence. The orientation is similar to that of subunit C–DNA interaction (panel (a)). The flipped Gua is on the edge of an open pocket. (g) The flipped Gua is surrounded by side chains of Trp92, Trp101 and Lys119 (which interacts with the 5′ phosphate). (h) Superimposition of 5mC-bound subunit C (in blue) and Gua-bound subunit D (in green) indicates that conformational changes (red arrows) occur between specific (yellow DNA) and non-specific interactions (gray DNA).

Figure 4.
Figure 4.

MspJI mutagenesis. (a) SDS-PAGE showing the deletion mutants used for activity. (b) Activities of MspJI WT and deletion mutants performed as 4-fold serial titrations (lanes 1–4). Approximately equal amount of purified enzymes was added into the starting point of each serial dilution. Based on the cleavage activity showed on the gel, we estimate that the decrease in activity for all the deletion mutants is greater than 64-fold. (c) Activity of K173F mutant.

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