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

Journal Article · · Scientific Reports
DOI:https://doi.org/10.1038/srep04246· OSTI ID:1253797
 [1];  [2];  [3];  [3];  [3];  [3];  [1];  [1];  [3];  [3];  [3];  [1]
  1. Emory Univ. School of Medicine, Atlanta, GA (United States)
  2. Sapphire Energy, San Diego, CA (United States); New England Biolabs, Ipswich, MA (United States)
  3. New England Biolabs, Ipswich, MA (United States)
+ Show Author Affiliations
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.
Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Inst. of Health; USDOE
Grant/Contract Number:
W-31109-ENG-38
OSTI ID:
1253797
Journal Information:
Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 4; ISSN 2045-2322
Publisher:
Nature Publishing GroupCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

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Cited By (8)

Structural basis for the recognition of sulfur in phosphorothioated DNA journal November 2018
Mechanistic insights into the recognition of 5-methylcytosine oxidation derivatives by the SUVH5 SRA domain journal February 2016
Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins journal January 2015
Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA journal June 2014
Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix journal September 2014
Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI journal May 2015
Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities journal April 2016
Chemical Display of Pyrimidine Bases Flipped Out by Modification-Dependent Restriction Endonucleases of MspJI and PvuRts1I Families journal December 2014

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59 BASIC BIOLOGICAL SCIENCES