/
Type II restriction enzymes searching for one site and then Type II restriction enzymes searching for one site and then

Type II restriction enzymes searching for one site and then - PowerPoint Presentation

natalia-silvester
natalia-silvester . @natalia-silvester
Follow
402 views
Uploaded On 2016-07-07

Type II restriction enzymes searching for one site and then - PPT Presentation

Stephen Halford DNAProteins Interactions Unit Department of Biochemistry Why study the enzymology of Type II restriction enzymes Enzyme specificity cf aminoacyl tRNA ID: 393883

site dna sfii ecorv dna site ecorv sfii restriction halford amp sites type specific protein endonuclease min enzymes kinetics

Share:

Link:

Embed:

Download Presentation from below link

Download Presentation The PPT/PDF document "Type II restriction enzymes searching fo..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.


Presentation Transcript

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 (6030 cm {hd}). 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