Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI

• Type of Material: Article
• Authors: John Horton, Emory University; Rebecca L. Nugent, New England Biolabs; Andrew Li, New England Biolabs; Megumu Yamada Mabuchi, New England Biolabs; Alexey Fomenkov, New England Biolabs; Devora Cohen-Karni, New England Biolabs; Rose M. Griggs, Emory University; Xing Zhang, Emory University; Geoffrey G. Wilson, New England Biolabs; Yu Zheng, New England Biolabs; Shuang-yong Xu, New England Biolabs; Xiaodong Cheng, Emory University
• Language: English
• Date: 2014
• Publisher: Nature Publishing Group: Open Access Journals - Option B
• Publication Version: Final Published Version
• Copyright Statement: © 2014, Macmillan Publishers Limited. All rights reserved
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://www.nature.com/srep/2014/140307/srep04246/full/srep04246.html
• Title of Journal or Parent Work: Scientific Reports
• ISSN: 2045-2322
• Volume: 4
• Start Page: 4246
• End Page: 4246
• Grant/Funding Information: X.C. is a Georgia Research Alliance Eminent Scholar; Use of the Advanced Photon Source was supported by the U.S. Department of Energy under Contract W-31-109-Eng-38.; New England Biolabs supported the mutagenesis work; National Institutes of Health (Grant GM049245-20) supported the crystallographic work
• Research Categories: Biology, Genetics

Abstract

The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3′ to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5′ to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.