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 specificityc.f. aminoacyl tRNA synthetases

DNA sequence recognitionc.f. cI and LacI repressors

Target site location along DNAc.f. Lac repressor, RNA polymerase

Test systems for DNA looping and synapsisc.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: 106 – 109

Aa tRNA synthetases: 103 – 104

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 3942Type III 22Type IV 18

Weirdos 1

Putative REs (in sequenced genomes) 21557

Nigel Brown, Biochemistry, Bristol, ~1980

416 (site 5)

421 (site 2)

Getting started on EcoRI, 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 (6030 cm {hd}). 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)

BfiI at:

ACTGGG(n5)TGACCC(n4)

EcoRV – now the archetype of the Type II restriction enzymesEcoRV – 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(n9)CCTAC(n13)

SfiI at:GGCCnnnnnGGCCCCGGnnnnnCCGG

BcgI at:

(n10)CGA(n6)TGC(n12)(n12)GCT(n6)ACG(n10)

SgrAI at:

CRCCGGYGGYGGCCRC+ 2 ( ± 1) Mg2+

per active site

What a difference a bp makes

C 0 10 20 30 40 50 60 min

0L

S

0 1 3 5 7 10 20 30 40 50 60 90 120 min

SXY

OL

1 unit EcoRV per µg DNA

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

pAT1533658 bp:

One EcoRV site

Taylor, J. D. & Halford, S. E. (1989). Discrimination between DNA sequences by the EcoRV restriction endonuclease. Biochemistry, 28, 6198-6207.

Only band seen with specific DNA when Ca2+ 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). EcoRV 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 32P-labelled DNA in EDTA-buffer (no Mg2+).

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 Mg2+ 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 [Mg2+]

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 CG: 24 bp

CC CC: 30 bp

CG CG: 30 bp

GCTGAGGCGACTCC

R2

R1

CCTCAGCGGAGTCG

CCTCAGCGGAGTCG

R2

R1

CCTCAGCGGAGTCG

R2

R1

R2

R1

R2

R1

Application of BbvCI to short-distance slidingApplication 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.

Direct evidence for “sliding” along DNA

Progressive reactions 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 MinicircleCatenane

Substrates to test for facilitated diffusion by EcoRV

Substrates to test for facilitated diffusion by EcoRV

Resolvase HindIII

EcoRV

H

R

REcoRV

H

3120 bp

346 bp

3466 bp

3120 bp

EcoRV

346 bp

Darren GowersGowers, 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

Partitioning of EcoRV on relaxed DNA:plasmid / catenane / minicircle

DN

A Pr

oduc

ts /

nM

0 10 20 30

Minicircle

Plasmid

0 10 20 30

4

8

12

Catenane

Minicircle

+ ++E

E

EE

E

E

0 10 20 30

Catenane

Plasmid

Time / min

Ratio: 1.1Ratio: 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 pointDissociation from DNA

BfiI at:

ACTGGG(n5)TGACCC(n4)

EcoRV – now the archetype of the Type II restriction enzymesEcoRV – 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(n9)CCTAC(n13)

SfiI at:GGCCnnnnnGGCCCCGGnnnnnCCGG

BcgI at:

(n10)CGA(n6)TGC(n12)(n12)GCT(n6)ACG(n10)

SgrAI at:

CRCCGGYGGYGGCCRC+ 2 Mg2+ per

active site

The SfiI restriction endonuclease

5’-G-G-C-C-n-n-n-nn-G-G-C-C -3’ 3’-C-C-G-G-nn-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 available

Stable protein (assayed at 50 C)

Already crystallised – crystals with Aneel Aggarwal

Time (min)0 30 60 90 120 150 180

Fina

l pro

duct

(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

DN

A (n

M)

0

1

2

3

4

5SC

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.

5 6 7 84 9 10 100 1 2 3C30 0

+ + + ++ + + -+ + + +SfiI -5 4 3 26 1 0 010 9 8 7C17 10

30-mer17-mer

SfiI (5nM) in Ca2+ 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 = 123,339MW from aa sequence:Monomer = 31,044Tetramer = 124,176

SfiI, a tetramer binding two DNA sitesRe

sidu

als

-1

0

1

Centrifugal radius5.90 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 transLooped 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 sites

EcoRV BglIBamHIEcoRI

NaeIEcoRIISau3AI

Type II(P)

Type IIE

BcgIAloIBaeIBplI

Type IIB

SfiINgoMIVCfr10ISgrAI

Type IIF

Type IIS

FokIBfiIBspMIMboII

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

DIGBIO

318 bp 237 bp554 bp

SfiI1 SfiI2

Anti-DIG coated glassDIG

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 Mg2+ - DNA looping and release

0 10 20 30 40 500

50

100

RMS

(nm

)

t (min)

150

200

250

300 0.5 sec filtered data trace Binary trace

# counts

tcc 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.

DNA release Unlooped DNA

Looped DNA

TPM records of loop capture and bead release

TPM: Native SfiI in Mg2+ - 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: ka = 2.108 M-1s-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 particleRapid reaction kinetics

Tethered particleKinetics

Tethered particleKinetics

Niels Laurens Gijs WuiteDave 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