Type II restriction enzymes
searching for one site and then two
Stephen Halford
DNA-Proteins Interactions Unit,
Department of Biochemistry,
Why study the enzymology of Type II restriction enzymes?
 Enzyme specificity
c.f. aminoacyl tRNA synthetases
DNA sequence recognition
Discrimination between alternative
(naturally-occurring) substrates:
Restriction enzymes: 106 – 109
Aa tRNA synthetases: 103 – 104
c.f. cI and LacI repressors
 Target site location along DNA
c.f. Lac repressor, RNA polymerase
 Test systems for DNA looping and synapsis
c.f. AraC, LacI, site-specific recombination
But much easier to measure the arrival of a Type II restriction
enzyme at its target sequence than a transcription factor:
Restriction enzyme - DNA gets cleaved at the recognition site
Transcription factor - level of gene expression gets modulated
Starting from ………..
Ph.D. (1967-70) and post-doc (1972-76)
with Freddie Gutfreund: Enzyme kinetics
and mechanisms – alkaline phosphatase,
lysosyme and -lactamase
Freddie in Cambridge,
1952 (long before moving
to Bristol), flanked by
colleagues from the
Cavendish Laboratory
Courtesy of the Cold Spring Harbor Laboratory Archives.
Restriction enzymes 1977 (all of them)
At http://rebase.neb.com,
October 2013
Enzymes
4087
Type I
Type II
Type III
Type IV
105
3942
22
18
Weirdos
1
Putative REs (in sequenced
genomes)
21557
Nigel Brown, Biochemistry, Bristol, ~1980
Getting started on EcoRI, with a
little help from Ken and Noreen ...
416
(site 5)
421
(site 2)
Halford, S. E., Johnson, N. P. & Grinsted, J. (1980). The EcoRI restriction
endonuclease with bacteriophage  DNA. Kinetic studies. Biochem. J. 191, 581-592.
Purification of the EcoRI restriction enzyme ~1978
1. At Centre for Applied Microbiology, Porton Down, grow 2  400 L
fermentor runs of Escherichia coli RY13 (the native strain for EcoRI).
2. Break open cells in a French press connected directly to a continuous
centrifuge and flow output into a bath tub.
3. Use overhead gantry to deposit sackful of DEAE cellulose into bathtub.
Marc Zabeau (then at EMBL. Previously with Rich Roberts, Cold Spring Harbor Laboratory)
Stir with oar. (EcoRI absorbs onto the DEAE).
4. Pump
contents ofstrain
bathtub
into the
drum ofprotein
a spin 
drier
linedinwith
Over-producing
for EcoRI
 insoluble
crystals
USA a
muslin bag. Spin hard to remove as much liquid as possible.
Over-producing
EcoRVbag
 soluble
protein
 to
crystal
structures
with
5. Deposit
contents strain
of thefor
muslin
into 0.2
M NaCl
release
the EcoRI.
Fritz Winkler (at EMBL)
Filter
to remove the DEAE cellulose.
6. Apply filtrate to P11 phosphocellulose column (6030 cm {hd}). Batchwash column with progressively increasing [NaCl]. (EcoRI elutes ~0.5 M
NaCl). Collect fractions in Winchester bottles.
7. Take the best two Winchesters back to Bristol for final “polishing”. End
up with ~10 ml at 30,000,000 units/ml.
EcoRV – now the archetype of the Type II restriction enzymes
BfiI at:
EcoRV at:
ACTGGG(n5)
TGACCC(n4)
5’--GATATC--3’
3’--CTATAG--5’
FokI at:
GGATG(n9)
CCTAC(n13)
SgrAI at:
CRCCGGYG
GYGGCCRC
+ 2 ( ± 1) Mg2+
per active site
5’-GAT
3’-CTA
ATC-3’
TAG-5’
SfiI at:
GGCCnnnnnGGCC
CCGGnnnnnCCGG
BcgI at:
(n10)CGA(n6)TGC(n12)
(n12)GCT(n6)ACG(n10)
What a difference a bp makes
C
1 unit EcoRV
per µg DNA
0
10 20 30 40 50 60 min
0
L
S
pAT153
3658 bp:
One EcoRV site
0 1 3 5 7 10 20 30 40 50 60 90 120 min
O
L
1 million units
EcoRV per µg
DNA
Ratio of EcoRV activities (kcat/Km values) at
recognition site (GATATC) over next best
site (GTTATC) = 1.106
S
X
Y
Taylor, J. D. & Halford, S. E. (1989). Discrimination between DNA sequences by the EcoRV
restriction endonuclease. Biochemistry, 28, 6198-6207.
EcoRV binds all DNA sequences with equal affinity
Gel-shifts with increasing concs EcoRV added to 0.1 nM 32P-labelled DNA in EDTA-buffer
(no Mg2+).
DNA – 381 bp with one EcoRV site
0 0.25 0.5 1
2 3 4 5 10 20 nM EcoRV
Same result with an 381 bp
DNA with no EcoRV site:
>15 retarded bands
With 50 bp DNA – 3 retarded bands
Only band seen
with specific
DNA when Ca2+
was added:
Vipond & Halford,
1995
With 100 bp DNA – 6 retarded bands
With 200 bp DNA – 12 retarded bands
Taylor, J. D., Badcoe, I. M., Clarke, A. R. & Halford, S. E. (1991). EcoRV restriction
endonuclease binds all DNA sequences with equal affinity. Biochemistry, 30, 8743-8753.
EcoRV binds Mg2+ only when at its cognate site
(A) EcoRV activity vs [Mg2+]
Vermote, C.L.M & Halford,S.E. (1992). EcoRV restriction
endonuclease: communication between catalytic metal ions
and DNA recognition. Biochemistry 31, 6082-6089.
(B) EcoRV bound to:
Specific DNA
Non-specific DNA
Winkler, F. K., et al. (1993). The crystal structure of EcoRV
endonuclease and of its complexes with cognate and non-cognate
DNA fragments. EMBO J. 12, 1781-1795.
von Hippel, P. H. & Berg, O. G. (1989) Facilitated target
location in biological systems. J. Biol. Chem., 264, 675 - 678.
3-D
1-D
Must be sliding because:
(i) Association rate very fast,
“too fast” for 3-D.
(ii) 1-D faster than 3-D.
A restriction enzyme at an asymmetric sequence (with Geoff Wilson)
EcoRV at a symmetrical site:
One gene – homodimer
5’-GATATC-3’
3’-CTATAG-5’
R gene
R2
BbvCI at an asymmetric site: 5’-CCTCAGC-3’
Two genes – heterodimer
3’-GGAGTCG-5’
R1
R2
R1 gene
R2 gene
R1
Heiter, D. F., Lunnen, K. D. & Wilson, G. G. (2005). Site-specific DNA-nicking mutants
of the heterodimeric restriction endonuclease R.BbvCI. J. Mol. Biol. 348, 631-640.
Application of BbvCI to short-distance sliding
1) Two BbvCI sites in direct repeat
R2
CCTCAGC
GGAGTCG
CCTCAGC
GGAGTCG
R1
R2
CC  CC: 30 bp
CG  CG: 30 bp
R1
Here, sites 30 bp apart.
Also made DNA with sites
40, 45 and 75 bp apart
2) Two BbvCI sites in inverted repeat
CC  CC: 30 bp
GCTGAGG
CGACTCC
CG CG: 24 bp
R1
R2
CCTCAGC
GGAGTCG
R1
R2
R1
R2
Gowers, D. M., Wilson, G. G. & Halford, S. E. (2005) Measurement of the contributions of 1D and 3D
pathways to the translocation of a protein along DNA. Proc. Natl. Acad. Sci .U.S.A. 102, 15883-15888.
Direct evidence for “sliding” along DNA
Progressive reactions that cut both BbvCI sites
(% total DNA cleavage reactions)
Sites separated by 30-45 bp
Sites separated by 75 bp
[NaCl]
0
60
150
46
33
40
42
29
25
23
22
15
15
13
13
But only over  45 bp at [NaCl]  60 mM
Substrates to test for facilitated
diffusion by EcoRV
Catenane
Plasmid
Minicircle
R
EcoRV
3466 bp
R
EcoRV
3120 bp
H
H
3120 bp
346 bp
EcoRV
Resolvase
HindIII
346 bp
Darren Gowers
Gowers, D. M. & Halford, S. E. (2003). Protein motion
from non-specific to specific DNA by three-dimensional
routes aided by supercoiling. EMBO J. 22, 1410-1418.
Partitioning of EcoRV on relaxed DNA:
plasmid / catenane / minicircle
E
E
DNA Products / nM
+
+
E
+
E
E
E
12
Catenane
Plasmid
8
Plasmid
4
Minicircle
0
10
20
30
Catenane
Minicircle
0
10
20
30
0
10
20
Time / min
Ratio: 3.4
Ratio: 2.6
Ratio = 14.0 on supercoiled DNA
Ratio: 1.1
30
Pathway to a specific DNA site
Initial random association
Sliding  50 bp at landing point
Dissociation
from DNA
Re-association
to new site in
same DNA
New landing site
close to rec. site
Sliding  50 bp at each
new landing point
Halford, S. E. & Marko, J. F. (2004). How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res., 32, 3040-3052.
Halford, S. E. (2009). An end to 40 years of mistakes in DNA-protein association kinetics? Biochem. Soc. Trans., 37, 343-348.
EcoRV – now the archetype of the Type II restriction enzymes
BfiI at:
EcoRV at:
ACTGGG(n5)
TGACCC(n4)
5’--GATATC--3’
3’--CTATAG--5’
FokI at:
GGATG(n9)
CCTAC(n13)
SgrAI at:
CRCCGGYG
GYGGCCRC
+ 2  Mg2+ per
active site
5’-GAT
3’-CTA
ATC-3’
TAG-5’
SfiI at:
GGCCnnnnnGGCC
CCGGnnnnnCCGG
BcgI at:
(n10)CGA(n6)TGC(n12)
(n12)GCT(n6)ACG(n10)
The SfiI restriction endonuclease
From Ira Schildkraut, New England Biolabs
5’-G-G-C-C-n-n-n-nn-G-G-C-C -3’
3’-C-C-G-G-nn-n-n-n-C-C-G-G -3’
8 bp recognition sequence – but interrupted by 5 bp nonspecific DNA
Over-producing strain available
Stable protein (assayed at 50 C)
Already crystallised – crystals with Aneel Aggarwal
Steady-state reactions of SfiI on one- and two-site DNA
(a) Two-site plasmid
Intact SC DNA
5
1 cut DNA
(b) Comparison of rates of formation of
final product from plasmids with 1 or
with 2 SfiI sites
5
2  cut
3
1  cut
2
1
0
Final product (nM)
SC
4
DNA (nM)
2  cut DNA
4
2-site DNA
3
2
1-site DNA
1
0
0
20
40
60
80 100 120
0
30
Time (min)
Wentzell, L. M., Nobbs, T. J. & Halford, S. E. (1995). The SfiI restriction endonuclease makes a fourstrand DNA break at two copies of its recognition sequence. J. Mol. Biol. 248, 581-595.
60
90
120 150 180
Time (min)
SfiI, a tetramer binding two DNA sites
Residuals
Equilibrium sedimentation:
Distribution of SfiI vs centrifugal
radius after 20 hrs at 10,000 rpm
Complexes with two DNA duplexes
SfiI (5nM) in Ca2+ binding buffer with:
+ 0  10 nM specific 30-mer
+ 10  0 nM specific 17-mer
Samples analysed on polyacrylamide gel
1
0
-1
A280
0.6
SfiI - + +
C17 10 10 9
C30 0 0 1
0.4
+
8
2
+
7
3
+
6
4
+
5
5
+
4
6
+
3
7
+
2
8
+
1
9
+
0
10
0
10
0.2
5.90
5.95
6.00
6.05
Centrifugal radius
30-mer
17-mer
MW from fit = 123,339
MW from aa sequence:
Monomer = 31,044
Tetramer = 124,176
Embleton, M. L., Williams, S. A., Watson, M. A. & Halford, S. E. (1999).
Specificity from the synapsis of DNA elements by the SfiI endonuclease.
J. Mol. Biol. 289, 785-797.
SfiI, a tetramer acting at two DNA sites
Initial model for SfiI on DNA with two and
with one recognition site(s)
SfiI with 2  GGCCnnnnnGGCC
Inactive T state
Active R state
Aneel Aggarwal
Two sites in cis
Looped DNA
Two sites in trans
Bridged DNA
Type IIS
Type II(P)
EcoRV
BamHI
EcoRI
Type IIE
LOOPS
FokI
BfiI
BspMI
MboII
BglI
Type IIB
LOOPS
BcgI
AloI
BaeI
BplI
Type IIF
LOOPS
NaeI
EcoRII
Sau3AI
LOOPS
Roberts,R.J.et al. (2003) A nomenclature for restriction enzymes, DNA
methyltransferases, homing endonucleases and their genes. Nucleic Acids Res.
31, 1805-1812.
SfiI
NgoMIV
Cfr10I
SgrAI
Tethered Particle Motion (TPM)
 Tracking the Brownian motion of a bead tethered by a DNA molecule, by video
microscopy
 Change in DNA length caused by trapping a loop changes the Brownian motion of
the bead
Unlooped
Looped
Record position of bead at 50 Hz (RMS)
Substrate for SfiI:
Streptavidincoated bead
SfiI1
BIOTIN
SfiI2
DIG
BIO
318 bp
554 bp
237 bp
DIG
Anti-DIG
coated glass
TPM: Inactive SfiI mutant with Mg2+ - DNA looping and release
0.5 sec filtered data trace
Binary trace
300
RMS (nm)
250
200
150
r
tcc
100
# counts
50
0
0
10
20
30
40
50
t (min)
c:
Time spent in unlooped state
waiting for the next looping event
kinetics for loop capture
r : Time spent in looped state
waiting for the next loop release
kinetics for loop breakdown
Laurens, N., Bellamy, S. R., Harms, A. F., Kovacheva, Y. S., Halford, S. E. & Wuite, G. J. (2009). Dissecting protein-induced DNA looping
dynamics in real time. Nucleic Acids Res. 37, 5454-5464.
TPM: Native SfiI in Mg2+ - DNA looping and cleavage
TPM records of loop capture and bead release
Fraction of non-cleaved tethers vs time:
Unlooped DNA
DNA release
½ for bead
release = 51 min
Looped DNA
From rapid-reaction kinetics of DNA cleavage by SfiI on the same two-site DNA:
E + S  E.S (at one site)  E.L (looped)  E.L  E.P  E + P
DNA binding:
ka = 2.108 M-1s-1
½ for DNA cleavage
= 0.05 min
½ for product
release = 60 min
DNA looping by SfiI: single molecules = bulk solution
Tethered particle
Rapid reaction kinetics
Tethered particle
Kinetics
Dave Rusling
Niels Laurens
Tethered particle
Kinetics
Gijs Wuite
Steve Halford’s lab reunion, 2011
From Tony Maxwell (1977-81)
and Rachel Smith (2008-13)
to Christian Pernstich (2006-13)
Mark Szczelkun: “The Halford Victims”
From commentary by John Widom on:
Gowers, D.M., Wilson,G.G & Halford,S.E. (2005) Measurement of the contributions of 1D
and 3D pathways to the translocation of a protein along DNA. PNAS, 102, 15883-15888.
Widom, J. (2005) PNAS 102,
16909-10.
The impossibility of such a
rotation can be appreciated by
imagining the protein to be a hot
dog bun lying over a hot dog. For
a hot dog oriented along the y
axis, rotation of the bun about
the x axis is forbidden because it
requires the bun to cross through
the dog.
©2005 by National Academy of Sciences
Direct evidence for “sliding” along DNA
NaCl
(mM)
BbvCI reactions that cut both sites:
(% total reactions)
30 bp (same at 40 or 45 bp)
Repeated /
Inverted sites
0
75 bp
Ratio
Repeated /
Inverted sites
Ratio
46 / 33
1.4
40 / 42
1
60
29 / 25
1.15
23 / 22
1
150
15 / 15
1
13 / 13
1
But only over  45 bp at [NaCl]  60 mM