1. Type II restriction enzymes searching for one site and then two Stephen Halford DNA-Proteins Interactions Unit, Department of Biochemistry,

2. 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

3. 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 ………..

4. Restriction enzymes 1977 (all of them) At http://rebase.neb.com, October 2013 Enzymes4087 Type I 105 Type II3942 Type III 22 Type IV18 Weirdos1 Putative REs (in sequenced genomes) 21557 Nigel Brown, Biochemistry, Bristol, ~1980

5. 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.

6. 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)

7. BfiI at: ACTGGG(n 5 ) TGACCC(n 4 ) EcoRV – now the archetype of the Type II restriction enzymes 5’-GATATC-3’ 3’-CTATAG-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 ( ± 1) Mg 2+ per active site

8. What a difference a bp makes C 0 10 20 30 40 50 60 min 0LS0LS 0 1 3 5 7 10 20 30 40 50 60 90 120 min S  X  Y  OL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 EcoRV restriction endonuclease. Biochemistry, 28, 6198-6207.

9. 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). 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 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

10. (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+ ]

11. 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.

12. 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 geneR2 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.

13. 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.

14. Direct evidence for “sliding” along DNA Progressive reactions that cut both BbvCI sites (% total DNA cleavage reactions) [NaCl] Sites separated by 30-45 bpSites separated by 75 bp 0 46334042 60 29252322 150 15 13 But only over  45 bp at [NaCl]  60 mM

15. PlasmidMinicircle Catenane Substrates to test for facilitated diffusion by EcoRV ResolvaseHindIII EcoRV H R R H 3120 bp 346 bp 3466 bp 3120 bp EcoRV 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.

16. Partitioning of EcoRV on relaxed DNA: plasmid / catenane / minicircle DNA Products / nM 0102030 Minicircle Plasmid 0102030 4 8 12 Catenane Minicircle +++ E E E E E E 0102030 Catenane Plasmid Time / min Ratio: 1.1 Ratio: 3.4 Ratio: 2.6 Ratio = 14.0 on supercoiled DNA

17. 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

18. BfiI at: ACTGGG(n 5 ) TGACCC(n 4 ) EcoRV – now the archetype of the Type II restriction enzymes 5’-GATATC-3’ 3’-CTATAG-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 site

19. 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 available  Stable protein (assayed at 50  C)  Already crystallised – crystals with Aneel Aggarwal

20. Time (min) 0306090120150180 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 Intact SC DNA 1  cut DNA2  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.

21. 5678 4 910 01 2 3C 30 0 ++++ + ++-++ + +SfiI- 5432 6 100109 8 7C 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 0 1 Centrifugal radius 5.905.956.006.05 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.

22. 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 sites

23. EcoRVBglI 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

24. DIG BIO 318 bp 237 bp 554 bp SfiI1SfiI2 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

25. TPM: Inactive SfiI mutant with Mg 2+ - DNA looping and release 01020304050 0 100 RMS (nm) t (min) 150 200 250 300 0.5 sec filtered data trace Binary trace # counts tctc cc r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.

26. 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:

27. 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

28. 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”

29. ©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. 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.

30. 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 046 / 331.440 / 421 6029 / 251.1523 / 221 15015 / 15113 / 131 Direct evidence for “sliding” along DNA But only over  45 bp at [NaCl]  60 mM