searching for one site and then two

sixcageyMechanics

Feb 22, 2014 (3 years and 1 month ago)

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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


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

Discrimination between alternative
(naturally
-
occurring) substrates:

Restriction enzymes: 10
6



10
9

Aa

tRNA

synthetases
: 10
3



10
4


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 ………..

Restriction enzymes 1977 (all of them)

At http://rebase.neb.com,
October 2013


Enzymes


4087


Type I



105

Type II



3942

Type III


22

Type IV



18


Weirdos


1


Putative REs (in sequenced
genomes)


21557

Nigel Brown, Biochemistry, Bristol, ~1980


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.

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.
Stir with oar. (
EcoRI

absorbs onto the
DEAE
).

4.
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.

5.
Deposit contents of the muslin bag into 0.2 M
NaCl

to release the
EcoRI
.
Filter to remove the
DEAE

cellulose.

6.
Apply filtrate to P11
phosphocellulose

column (60

30 cm {
h

d
}). Batch
-
wash 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.


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)



Bfi
I

at:


ACTGGG
(n
5
)

TGACCC
(n
4
)

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
)


SgrA
I

at:


CRCCGGYG

GYGGCCRC

+ 2 (
±

1)
Mg
2+
per active site

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.

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 bands

(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+
]

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.

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.

CG


C䜺
24 bp

CC


䍃㨠
30 bp

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


䍃㨠
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.

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 mM

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.

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 DNA

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 DNA


BfiI

at:


ACTGGG
(n
5
)

TGACCC
(n
4
)

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



2+
per
active site

The SfiI restriction endonuclease

5’
-
G
-
G
-
C
-
C
-
n
-
n
-
n
-
n

n
-
G
-
G
-
C
-

-
3’

3’
-
C
-
C
-
G
-
G
-
n

n
-
n
-
n
-
n
-
C
-
C
-
G
-

-
3’

From Ira
Schildkraut
, New England
Biolabs


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

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


ct



2


ct

Intact SC DNA

1


捵琠䑎D




捵琠
䑎D

(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.

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.

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


䝇䍃湮湮湇䝃C

Aneel Aggarwal

SfiI, a tetramer acting at two DNA sites

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

LOOPS

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

Looped

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






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
.

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⹌†


E⹐.


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:

DNA looping by SfiI: single molecules = bulk solution

Tethered particle

Rapid reaction kinetics

Tethered particle



Kinetics

Tethered particle

Kinetics

Niels

Laurens

Gijs

Wuite

Dave
Rusling

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”

©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.

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