Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease

• Type of Material: Article
• Authors: John Horton, Emory University; Megumu Yamada Mabuchi, New England Biolabs Inc; Devora Cohen-Karni, New England Biolabs Inc; Xing Zhang, Emory University; Rose M. Griggs, Emory University; Mala Samaranayake, New England Biolabs Inc; Richard J. Roberts, New England Biolabs Inc; Yu Zheng, New England Biolabs Inc; Xiaodong Cheng, Emory University
• Language: English
• Date: 2012-10-01
• Publisher: Oxford University Press (OUP): Policy C - Option B
• Publication Version: Final Published Version
• Copyright Statement: © 2012 Horton et al. Published by Oxford University Press
• License: Creative Commons Attribution-Noncommercial 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1093/nar/gks719
• Title of Journal or Parent Work: Nucleic Acids Research
• ISSN: 0305-1048
• Volume: 40
• Issue: 19
• Start Page: 9763
• End Page: +
• Grant/Funding Information: Funding for open access charge: NIH.; The U.S. National Institutes of Health (NIH) (GM095209; to Y.Z. and GM049245-18 to X.C.) and New England Biolabs. X.C. is a Georgia Research Alliance Eminent Scholar.; The Department of Biochemistry at the Emory University School of Medicine supported the use of the Southeast Regional Collaborative Access Team synchrotron beamlines at the Advanced Photon Source of Argonne National Laboratory.
• Keywords: MECHANISM; Life Sciences & Biomedicine; Science & Technology; FOKI; HEMI-METHYLATED DNA; SRA DOMAIN; FAMILY; UHRF1; 5-METHYLCYTOSINE; BINDING DOMAIN; RECOGNITION; Biochemistry & Molecular Biology; MAMMALIAN DNA
• Research Categories: Chemistry, Biochemistry

Abstract

The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N12/N16) away from the modified cytosine at the 30-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-toback dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N12 cleavage in the same strand and then the second distal N16 cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans. © 2012 The Author(s).