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

Horton JR, Wang H, Mabuchi MY, Zhang X, Roberts RJ, Zheng Y, Wilson GG, Cheng X - Nucleic Acids Res. (2014)

Bottom Line: We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.

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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.
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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.

Mentions: We crystallized MspJI in the presence of a 27-bp asymmetric dsDNA oligo containing the hemimethylated recognition site, 5mCGGG, close to the 5′ end of the top strand. Previous experiments indicated that this was the shortest oligo MspJI could cleave. The structure was solved by molecular replacement and refined to a resolution of 3.0 Å (Table 1). The crystallographic asymmetric unit contained one MspJI tetramer and one DNA duplex (Figure 1a), even though crystallization was carried out under conditions of two duplexes per tetramer. This discrepancy stemmed from crystal packing. Although each tetramer interacted with two symmetry-related DNA molecules (Figure 1b and c), every DNA molecule also interacted with two symmetry-related tetramers, a consequence of crystallization that reduced the overall stoichiometry to 1:1 (Figure 1d). The proximal portion of each duplex, containing the methylated MspJI recognition sequence (Figure 1e), interacted specifically with the DNA-binding domain of subunit C of one tetramer, and the distal portion interacted non-specifically with the DNA-binding domain of subunit D of the symmetry-related tetramer (Figure 1f), mediating crystal lattice interactions along the crystallographic c-axis (Figure 1a).


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

Horton JR, Wang H, Mabuchi MY, Zhang X, Roberts RJ, Zheng Y, Wilson GG, Cheng X - Nucleic Acids Res. (2014)

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.
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Related In: Results  -  Collection

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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.
Mentions: We crystallized MspJI in the presence of a 27-bp asymmetric dsDNA oligo containing the hemimethylated recognition site, 5mCGGG, close to the 5′ end of the top strand. Previous experiments indicated that this was the shortest oligo MspJI could cleave. The structure was solved by molecular replacement and refined to a resolution of 3.0 Å (Table 1). The crystallographic asymmetric unit contained one MspJI tetramer and one DNA duplex (Figure 1a), even though crystallization was carried out under conditions of two duplexes per tetramer. This discrepancy stemmed from crystal packing. Although each tetramer interacted with two symmetry-related DNA molecules (Figure 1b and c), every DNA molecule also interacted with two symmetry-related tetramers, a consequence of crystallization that reduced the overall stoichiometry to 1:1 (Figure 1d). The proximal portion of each duplex, containing the methylated MspJI recognition sequence (Figure 1e), interacted specifically with the DNA-binding domain of subunit C of one tetramer, and the distal portion interacted non-specifically with the DNA-binding domain of subunit D of the symmetry-related tetramer (Figure 1f), mediating crystal lattice interactions along the crystallographic c-axis (Figure 1a).

Bottom Line: We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.

Show MeSH