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Author Notes:

Tel: +1 404 727 8491; Fax: +1 404 727 3746; Email: xcheng@emory.edu

J.R.H. performed crystallographic work; H.W. and M.Y.M. performed mutagenesis and activity assays; G.G.W. performed structural analysis and assisted in preparing the manuscript; X.C., X.Z., Y.Z. and R.J.R. organized and designed the scope of the study; and all were involved in analyzing data and preparing the manuscript.

The authors thank Don Comb and Jim Ellard for enlightened support for the research presented here; Brenda Baker and John Buswell of the Organic Synthesis group for DNA oligonucleotides; and Devora Cohen-Karni for involvement in the early stage of the project; and together with other members of the restriction enzymes and epigenetics research groups, for numerous thoughtful discussions. X.C. is a Georgia Research Alliance Eminent Scholar.

Conflict of interest statement. None declared.

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Research Funding:

The U.S. National Institutes of Health [GM095209, GM105132 to Y.Z.; GM049245-21 to X.C.]; New England Biolabs; Department of Biochemistry, Emory University School of Medicine, supports the use of Southeast Regional Collaborative Access Team (SER-CAT) beamline at the Advanced Photon Source (APS), Argonne National Laboratory; Use of the APS was supported by the U.S. department of Energy, Office of Science.

Funding for open access charge: New England Biolabs.

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

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Journal Title:

Nucleic Acids Research

Volume:

Volume 42, Number 19

Publisher:

, Pages 12092-12101

Type of Work:

Article | Final Publisher PDF

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.

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© The Author(s) 2014.

This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits making multiple copies, distribution of derivative works, distribution, public display, and publicly performance, provided the original work is properly cited. This license requires credit be given to copyright holder and/or author, copyright and license notices be kept intact.

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