Structural and Functional Analysis of the Engineered
Type I DNA Methyltransferase EcoR124I
NT
James E. Taylor
1
, Phil Callow
2
, Anna Swiderska
1
and G. Geoff Kneale
1
⁎
1
Biophysics Laboratories,
Institute of Biomedical and
Biomolecular Sciences,
University of Portsmouth,
Portsmouth PO1 2DT, UK
2
Partnership for Structural
Biology, Institut Laue Langevin,
38042 Grenoble Cedex 9,
Grenoble, France
Received 26 January 2010;
received in revised form
2 March 2010;
accepted 4 March 2010
Available online
17 March 2010
The Type I R–M system EcoR124I is encoded by three genes. HsdM is
responsible for modification (DNA methylation), HsdS for DNA sequence
specificity and HsdR for restriction endonuclease activity. The trimeric
methyltransferase (M
2
S) recognises the asymmetric sequence (GAAN
6-
RTCG). An engineered R–M system, denoted EcoR124I
NT
, has two copies
of the N-terminal domain of the HsdS subunit of EcoR124I, instead of a single
S subunit with two domains, and recognises the symmetrical sequence
GAAN
7
TTC. We investigate the methyltransferase activity of EcoR124I
NT
,
characterise the enzyme and its subunits by analytical ultracentrifugation and
obtain low-resolution structural models from small-angle neutron scattering
experiments using contrast variation and selective deuteration of subunits.
© 2010 Elsevier Ltd. All rights reserved.
Edited by K. Morikawa
Keywords: DNA methyltransferase; DNA methylation; restriction–modification;
sedimentation velocity; small-angle neutron scattering
Introduction
Restriction–modification (R–M) enzymes provide
a bacterial defence mechanism against foreign DNA.
Hemi-methylated host DNA is fully methylated at
specific sequences by a methyltransferase (MTase),
thus protecting its DNA from restriction by the
accompanying endonuclease (ENase). Foreign DNA
is unmethylated at these sites and is cleaved.
1,2
TypeI R–Msystems are hetero-oligomeric enzymes
encoded by three hsd (host specificity of DNA) genes
encoding three polypeptides: HsdS, responsible for
DNA recognition, HsdM for DNA modification and
HsdR for cleavage. The ENase requires all three
subunits: M,S and R while the MTaserequires just the
M and S subunits. For enzyme activity, the MTase is
dependent upon S-adenosylmethionine, while the
ENase in addition requires Mg
2+
and ATP. All Type I
R–M systems methylate a specific adenine at the N
6
position (for reviews, see Ref. 3).
DNA sequence alignments of the S subunits of
Type I R–M systems have shown the presence of two
variable regions that form target recognition
domains (TRDs), each recognising one-half of the
bipartite DNA recognition motif, and two conserved
regions that are believed to interact with the M
subunit. The DNA recognition sequence of Type I
R–M systems is, in general, asymmetric, and the two
TRDs within the S subunit have different amino acid
sequences. Typically, each of the DNA sequence
half-sites is 3–5bpinlength,separatedbya
nonspecific spacer region (5–8 bp). On the basis of
internal sequence homologies, a circular arrange-
ment of the domains of the S subunit was suggested,
which brings the N- and C-termini into close
proximity.
4
Circular permutations of the sequence
of the N- and C-terminal conserved regions of the S
subunit of EcoAI support this notion.
5
Crystal structures have been reported for the Type
I S subunits of Mycoplasma genitalium [Protein Data
Bank (PDB) code: 1YDX] and Methanococcus jan-
naschii (PDB code: 1YF2), confirming the proximity
of the N- and C-termini and in each case showing a
hetero-dimeric structure held together by the inter-
action of coiled-coil regions.
6,7
Crystal structures are
also available in the protein structure database for
*Corresponding author. E-mail address:
geoff.kneale@port.ac.uk.
Abbreviations used: R–M, restriction–modification;
MTase, methyltransferase; ENase, endonuclease; TRD,
target recognition domain; PDB, Protein Data Bank; ocr,
overcome classical restriction; SANS, small-angle neutron
scattering; SV, sedimentation velocity; SE, sedimentation
equilibrium; DLS, dynamic light scattering.
doi:10.1016/j.jmb.2010.03.008 J. Mol. Biol. (2010) 398, 391–399
Available online at www.sciencedirect.com
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.