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Author Notes:

E-mail Address : xcheng@emory.edu Tel: +1 404 727 8491 Fax: +1 404 727 3746 The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.

J.R.H. performed crystallographic work and gel filtration chromatography, J.G.B. and A.Q. performed site-directed mutagenesis, R.M.G. and X.Z. performed initial purification and crystallization trials, X.Z. suggested the hydrodynamic experiment, S.G. duplicated experiments on mutant activities and tested AbaSI activity on 5hmU, G.G.W. performed structural analysis and assisted in preparing the manuscript, X.C., Y.Z. and Z.Z. 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; Richard J. Roberts and Bill Jack for leadership and encouragement, and together with other members of the restriction enzymes and epigenetics research groups, for numerous thoughtful discussions.

We thank John Buswell and his Organic Synthesis group for DNA oligonucleotides; and Hideharu Hashimoto for help with DNA-binding assays. X.C. is a Georgia Research Alliance Eminent Scholar.

Conflict of interest statement. AbaSI and others restriction enzymes mentioned in this article are products of New England Biolabs, Inc., a company that studies, purifies and sells restriction enzymes.

The authors declare no competing interests.


Research Funding:

National Institutes of Health (NIH) [GM049245-20 to X.C.]; NIH Small Business Innovation Research [GM096723 to Z.Z.];

Emory University School of Medicine, Department of Biochemistry, supports the use of Southeast Regional Collaborative Access Team (SER-CAT) 22-ID 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: NIH.

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA


Journal Title:

Nucleic Acids Research


Volume 42, Number 12


, Pages 7947-7959

Type of Work:

Article | Final Publisher PDF


AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ~70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ~22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

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

This is an Open Access work distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/).

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