Stephen Halford DNAProteins Interactions Unit Department of Biochemistry Why study the enzymology of Type II restriction enzymes Enzyme specificity cf aminoacyl tRNA ID: 393883
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Slide1
Type II restriction enzymes searching for one site and then two
Stephen
Halford
DNA-Proteins Interactions Unit,
Department of Biochemistry,Slide2
Why study the
enzymology
of Type II restriction enzymes?
Enzyme specificity c.f. aminoacyl
tRNA synthetasesDNA sequence recognition
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 recombinationBut 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
Discrimination between alternative (naturally-occurring) substrates:
Restriction enzymes: 10
6
– 10
9
Aa
tRNA
synthetases
: 10
3
– 10
4
Slide3
Courtesy of the Cold Spring
Harbor
Laboratory Archives.
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
Starting from ………..Slide4
Restriction enzymes 1977 (all of them)
At http://rebase.neb.com, October 2013
Enzymes 4087
Type I 105
Type II 3942
Type III 22Type IV 18
Weirdos
1Putative REs (in sequenced genomes) 21557
Nigel Brown, Biochemistry, Bristol, ~1980Slide5
416 (site 5)
421 (site 2)
Getting started on
Eco
RI
, with a little help from Ken and Noreen ...
Halford, S. E., Johnson, N. P. &
Grinsted
, J. (1980). The EcoRI
restriction endonuclease with bacteriophage DNA. Kinetic studies. Biochem
. J.
191
,
581-592.Slide6
Purification of the
EcoRI
restriction enzyme ~1978
At
Centre for Applied Microbiology, Porton Down, grow 2 400 L fermentor
runs of Escherichia coli RY13 (the native strain for EcoRI).
Break open cells in a French press connected directly to a continuous centrifuge and flow output into a bath tub.
Use overhead gantry to deposit sackful of
DEAE cellulose into bathtub. Stir with oar. (EcoRI absorbs onto the
DEAE).Pump contents of bathtub into the drum of a spin drier lined with a muslin bag. Spin hard to remove as much liquid as possible.
Deposit contents of the muslin bag into 0.2 M
NaCl
to release the
EcoRI. Filter to remove the DEAE cellulose.Apply filtrate to P11 phosphocellulose column (6030 cm {hd}). Batch-wash column with progressively increasing [
NaCl
]. (
EcoRI
elutes ~0.5 M
NaCl
). Collect fractions in Winchester bottles.
Take the best two Winchesters back to Bristol for final “polishing”. End up with ~10 ml at 30,000,000 units/ml.
Marc
Zabeau
(then at EMBL. Previously with Rich Roberts, Cold Spring
Harbor
Laboratory)
Over-producing strain for
EcoRI
insoluble protein crystals in USA
Over-producing strain for
EcoRV
soluble protein crystal structures with Fritz Winkler (at EMBL)Slide7
Bfi
I
at:
ACTGGG(n5)
TGACCC(n4
)
EcoRV
– now the
archetype
of the Type
II restriction
enzymes
5
’
-GAT ATC-3’
3’-CTA TAG-5’
EcoRV
at:
5
’-
-GATATC--3’
3’--CTATAG--5’
FokI at:
GGATG
(n
9
)
CCTAC
(n
13
)
SfiI at:
GGCCnnnnnGGCC
CCGGnnnnnCCGG
BcgI at:
(n
10
)
CGA
(n
6
)
TGC
(n
12
)
(n
12
)
GCT
(n
6
)
ACG
(n10)
SgrA
I
at:CRCCGGYGGYGGCCRC
+ 2 (
± 1)
Mg2+ per active siteSlide8
What a difference a
bp
makes
C 0 10 20 30 40 50 60 min
0
L
S
0 1 3 5 7 10 20 30 40 50 60 90 120 min
S
X
Y
O
L
1 unit
EcoRV
per µg DNA
1
million
units
EcoRV
per µg DNA
Ratio of
EcoRV
activities (
k
cat
/
K
m
values) at recognition site (
GATATC
) over next best site (
GTTATC
) =
1.10
6
pAT153
3658
bp
:
One
EcoRV
site
Taylor, J. D. & Halford, S. E. (1989). Discrimination between DNA sequences by the
Eco
RV
restriction endonuclease.
Biochemistry,
28
, 6198-6207.Slide9
Only band seen with specific DNA when Ca
2+
was added:Vipond & Halford, 1995
0 0.25 0.5 1 2 3 4 5 10 20 nM
EcoRV
Taylor, J. D.,
Badcoe
, I. M., Clarke, A. R. & Halford, S. E. (1991).
Eco
RV
restriction endonuclease binds all DNA sequences with equal affinity.
Biochemistry,
30, 8743-8753.EcoRV binds all DNA sequences with equal affinity
Gel-shifts with increasing
concs
EcoRV
added to 0.1 nM
32
P-labelled DNA in
EDTA
-buffer (no Mg
2+
).
DNA – 381
bp
with one
EcoRV
site
With 50
bp
DNA
– 3 retarded bands
With 100
bp
DNA – 6 retarded bands
With 200
bp
DNA – 12 retarded bands
Same result with an 381
bp
DNA with no
EcoRV
site:
>15 retarded bandsSlide10
(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.
EcoRV
binds Mg
2+
only when at its cognate site
Vermote
, C.L.M &
Halford,S.E
. (1992).
EcoRV
restriction endonuclease: communication between catalytic metal ions and DNA recognition.
Biochemistry
31,
6082-6089.
(A)
EcoRV
activity
vs
[Mg
2+
]Slide11
von
Hippel
, P
. H
. & Berg, O. G. (1989) Facilitated target location in biological systems.
J. Biol. Chem., 264, 675 - 678
.
1-D
3-D
Must be sliding because:
(
i
) Association rate
very fast, “too fast” for 3-D.(ii) 1-D faster than 3-D.Slide12
A restriction enzyme
at
an asymmetric sequence (with Geoff Wilson)
BbvCI
at an asymmetric
site: 5’-CCTCAGC-3’
Two genes – heterodimer
3’-GGAGTCG-5
’
R2
R1
R1 gene
R2 gene
R gene
EcoRV
at a symmetrical site:
5’-GATATC-3’
One gene –
homodimer
3
’-
CTATAG-5’
R2
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.Slide13
CG
CG:
24 bp
CC
CC:
30 bp
CG
CG
: 30 bp
GCTGAGG
CGACTCC
R2
R1
CCTCAGC
GGAGTCG
CCTCAGC
GGAGTCG
R2
R1
CCTCAGC
GGAGTCG
R2
R1
R2
R1
R2
R1
Application of
BbvCI
to short-distance sliding
CC
CC:
30 bp
1) Two BbvCI sites in
direct
repeat
2) Two BbvCI sites in
inverted
repeat
Here, sites 30
bp
apart.
Also made DNA with sites 40, 45 and 75
bp
apart
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.Slide14
Direct evidence for “sliding” along DNA
Progressive
r
eactions
that cut both
BbvCI
sites
(%
total DNA cleavage
reactions)
[
NaCl
]
Sites separated by 30-45
bp
Sites separated by 75
bp
0
46
33
40
42
60
29
25
23
22
150
15
15
13
13
But only over
45
bp
at [
NaCl
] 60 mMSlide15
Plasmid
Minicircle
Catenane
Substrates to test for facilitated diffusion by
Eco
RV
Resolvase
Hin
dIII
Eco
RV
H
R
R
Eco
RV
H
3120 bp
346 bp
3466 bp
3120 bp
Eco
RV
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.Slide16
Partitioning of EcoRV on relaxed DNA:
plasmid / catenane / minicircle
DNA Products / nM
0
10
20
30
Minicircle
Plasmid
0
10
20
30
4
8
12
Catenane
Minicircle
+
+
+
E
E
E
E
E
E
0
10
20
30
Catenane
Plasmid
Time / min
Ratio:
1.1
Ratio:
3.4
Ratio:
2.6
Ratio = 14.0 on supercoiled DNASlide17
Re-association to new site in same DNA
Sliding
50
bp
at each new landing point
New landing site close to rec. site
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.
Pathway to a specific DNA site
Initial random association
Sliding
50
bp
at landing point
Dissociation from DNASlide18
BfiI
at:
ACTGGG
(n5)
TGACCC(n4)
EcoRV
– now the
archetype
of the Type
II restriction
enzymes
5
’
-GAT ATC-3’
3’-CTA TAG-5’
EcoRV
at:
5
’-
-GATATC--3’
3’--CTATAG--5’
FokI at:
GGATG
(n
9
)
CCTAC
(n
13
)
SfiI at:
GGCCnnnnnGGCC
CCGGnnnnnCCGG
BcgI at:
(n
10
)
CGA
(n
6
)
TGC
(n
12
)
(n
12
)
GCT
(n
6
)
ACG
(n
10
)
SgrAI
at:CRCCGGYG
GYGGCCRC
+ 2
Mg
2+
per active siteSlide19
The SfiI restriction endonuclease
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’
From Ira Schildkraut, New England
Biolabs
8
bp
recognition sequence – but interrupted by 5
bp nonspecific DNA Over-producing strain availableStable protein (assayed at 50 C)Already crystallised – crystals with Aneel AggarwalSlide20
Time (min)
0
30
60
90
120
150
180
Final product (nM)
0
1
2
3
4
5
1-site DNA
2-site DNA
(b) Comparison of rates of formation of final product from plasmids with 1 or with 2 SfiI sites
Steady-state reactions of SfiI on one- and two-site DNA
Time (min)
0
20
40
60
80
100
120
DNA (nM)
0
1
2
3
4
5
SC
1
cut
2
cut
Intact SC DNA
1
cut DNA
2
cut
DNA
(a) Two-site plasmid
Wentzell
, L. M.,
Nobbs
, T. J. & Halford, S. E. (1995). The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence.
J. Mol. Biol.
248
,
581-595.Slide21
5
6
7
8
4
9
10
10
0
1
2
3
C
30
0
+
+
+
+
+
+
+
-
+
+
+
+
SfiI
-
5
4
3
2
6
1
0
0
10
9
8
7
C
17
10
30-mer
17-mer
SfiI (
5nM
) in
Ca
2
+
binding buffer with:
+ 0
10 nM
specific
30-mer
+ 10
0 nM
specific
17-mer
Samples analysed on
polyacrylamide
gel
Complexes with two DNA duplexes
MW from fit = 123,339
MW from aa sequence:
Monomer = 31,044
Tetramer = 124,176
SfiI, a tetramer binding two DNA sites
Residuals
-1
0
1
Centrifugal radius
5.90
5.95
6.00
6.05
0.2
0.4
0.6
A
280
Equilibrium sedimentation:
Distribution of SfiI vs centrifugal radius after 20 hrs at 10,000 rpm
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.Slide22
Active R state
Inactive T state
Two sites
in cis
Two sites
in trans
Looped DNA
Bridged DNA
Initial model for SfiI on DNA with two and with one recognition site(s)
SfiI with 2
GGCCnnnnnGGCC
Aneel Aggarwal
SfiI, a tetramer acting at two DNA sitesSlide23
EcoRV
BglI
BamHI
EcoRI
NaeI
EcoRII
Sau3AI
Type II(P)
Type IIE
BcgI
AloI
BaeI
BplI
Type IIB
SfiI
NgoMIV
Cfr10I
SgrAI
Type IIF
Type IIS
FokI
BfiI
BspMI
MboII
Roberts,R.J
.et al
. (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.
Nucleic Acids
Res.
31
, 1805-1812
.
LOOPS
LOOPS
LOOPS
LOOPSSlide24
DIG
BIO
318
bp
237
bp
554 bp
SfiI1
SfiI2
Anti-DIG coated glass
DIG
BIOTIN
Streptavidin
-coated bead
Substrate for SfiI:
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
Tethered Particle Motion (
TPM
)
Record position of bead at 50 Hz (
RMS
)
Unlooped
LoopedSlide25
TPM
:
Inactive SfiI mutant with
Mg
2+
- DNA looping and release
0
10
20
30
40
50
0
50
100
RMS (nm)
t (min)
150
200
250
300
0.5 sec filtered data trace
Binary trace
# counts
t
c
c
r
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
.Slide26
DNA release
Unlooped DNA
Looped DNA
TPM records of loop capture and bead release
TPM
: Native SfiI
in
Mg
2+
- DNA looping and cleavage
½
for bead release =
51
min
Fraction of non-cleaved tethers
vs
time:
½
for product release = 60 min
E +
S
E.S (at one site)
E.L (looped)
E.L
E.P
E
+ P
½
for
DNA cleavage
=
0.05 min
DNA binding:
k
a
=
2.10
8
M
-1
s
-1
From rapid-reaction kinetics of DNA cleavage by SfiI on the same two-site DNA:Slide27
DNA looping by SfiI: single molecules = bulk solution
Tethered particle
Rapid reaction kinetics
Tethered particle
Kinetics
Tethered particle
Kinetics
Niels
Laurens
Gijs
Wuite
Dave
RuslingSlide28
From Tony Maxwell (1977-81) to Christian
Pernstich
(2006-13)
and Rachel Smith (2008-13)
Steve
Halford’s
lab reunion, 2011
Mark Szczelkun: “The Halford Victims”Slide29
©2005 by National Academy of Sciences
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.
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
.Slide30
NaCl
(mM)
BbvCI
reactions
that cut both sites:(% total reactions)
30
bp
(
same at 40
or
45
bp
)
75
bp
Repeated
/
Inverted sites
Ratio
Repeated
/
Inverted sites
Ratio
0
46
/
33
1.4
40
/
42
1
60
29
/
25
1.15
23
/
22
1
150
15
/
15
1
13
/
13
1
Direct evidence for “sliding” along DNA
But only over
45
bp
at [
NaCl
] 60 mM