1. Type II Restriction Enzymes, more than just cloning tools Alistair J. Jacklin, Yana S. Kovacheva, Jacqui J.T. Marshall, Christian Pernstich, Fiona L. Preece, Susan E. Retter, David A. Rusling, Kelly L. Sanders and Stephen E. Halford* The DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom Type II Restriction Enzymes EcoRV HindIII EcoRI PvuII NaeI EcoRII Type IIP Type IIE BcgI BaeI BplI Type IIB SfiI NgoMIV SgrAI Type IIF Type IIS FokI BspMI Fig. 2 – Some sub-types of Type II REs. Top row, varied cleavage positions relative to recognition site. Bottom row, varied reaction mechanisms. Type II REs Of the various types of RM systems, Type II enzymes are the best studied and their REs are commercially important. The classical Type II REs (Type IIP) recognise 4 – 8 bp sequences and cleave within the site. For example EcoRV cuts the sequence as shown by the arrows: Other Type II REs differ from Type IIP in a number of ways (Fig. 2). The Type IIS and Type IIB enzymes cut at specified positions away from their recognition sites. Some REs act at two sites: the Type IIE enzymes use one site to activate cleavage of the other site while the Type IIF enzymes cut two sites at the same time. RM systems are present in prokaryotes, where they protect the cell from incoming foreign DNA, particularly phage DNA. RM systems have two components, the restriction endonuclease (RE) and the methyltransferase (MT). The RE recognises specific sites in DNA and proceeds to cleave them in the presence of Mg 2+ (Fig. 1), unless the MT had previously protected those sites by methylating adenines or cytosines. The host DNA is methylated directly after replication and is thus protected against the RE. Restriction-Modification (RM) Systems Me M Hemi-methylated host DNA Foreign DNA (e.g. phage DNA) Methylated host DNA Cleavage by RE Methylation by MT Protection from RE Fig. 1 – How RM systems work. 5' -G-A-T A-T-C-3' 3' -C-T-A T-A-G-5' FokI Fig. 3 – Crystal structure of FokI bound to its recognition sequence. The Type IIS enzyme, FokI cleaves DNA at fixed positions downstream of an asymmetric sequence: It exists as a monomer in solution, with separate domains for DNA binding and catalysis (Fig. 3). However, the catalytic domain has one active site so can cut only one DNA strand. To cut both strands, two monomers have to associate via their catalytic domains to give a dimer with two active sites. DNA binding domain Catalytic domain Cleavage sites The SfiI RE, the archetype of the Type IIF systems (Fig.2), has a discontinuous recognition sequence, 5'-G-G-C-C-n-n-n-n n-G-G-C-C-3' 3'-C-G-C-C-n n-n-n-n-C-C-G-G-5 It exists as a tetramer and binds cooperatively two specific DNA duplexes. SfiI then cuts both duplexes before dissociating from the DNA. In the structure (Fig. 4), two subunits form one DNA-binding cleft on one side of the protein and the other two subunits a second cleft on the opposite side. Fig. 4 – Crystal structure of SfiI bound to two DNA duplexes. The Type IIB RE BcgI cleaves on both sides of its recognition site, a bipartite discontinuous sequence. It thus excises the site from the remainder of the DNA on a 32 bp fragment: BcgI contains two subunits, BcgIA and BcgIB, in a 2:1 ratio. Type II RM systems usually feature separate MT and RE proteins, but BcgIA carries both functions while BcgIB confers DNA sequence specificity. [The methylation state of the DNA determines whether cleavage or methylation occurs.] BcgI acts concertedly on plasmids with two sites, cleaving 8 phosphodiester bonds per reaction, but the A 2 B 1 protomer has only two nuclease sites. BcgI seems to need oligomeric forms of the protomer for its reactions. 5 - n-n n 10 -C-G-A-n-n-n-n-n-n-T-G-C-n 10 -n-n '3 3' n-n-n 10 -G-C-T-n-n-n-n-n-n-A-C-G-n 10 n-n - '5 The Type IIS enzyme, BspMI cleaves DNA at fixed positions downstream of an asymmetric sequence: Like FokI, BspMI has separate DNA-binding and catalytic domains but, in contrast to FokI, BspMI is a tetramer in solution, with four DNA-binding and four catalytic domains. Yet BspMI has no activity when bound to its recognition site. It becomes active only when bound to two cognate sites. It then proceeds to cut both strands at both sites in a single event. The tetrameric structure provides the 4 catalytic domains needed to cut the 4 bonds but seemingly an excess of DNA-binding domains. 5-A-C-C-T-G-C-n-n-n-n n-n-n-n-n-3' 3-T-G-G-A-C-G-n-n-n-n-n-n-n-n n-5 5-G-G-A-T-G-n-n-n-n-n-n-n-n-n n-n-n-n-n-3' 3-C-C-T-A-C-n-n-n-n-n-n-n-n-n-n-n-n-n n-5' BcgI BspMI SfiI Experimental Techniques Fig. 7 – Diffraction pattern of an initial crystal of BcgI (resolution 7.6 Å). Fig. 9 - AUC data for a low conc of BcgI. Theoretical A 2 B MW = 182kDa Fig. 5 – Schemes for the reactions of tetrameric SfiI (light blue circles) on a DNA duplex with the recognition sequence (red box, * marks the radiolabel): (A), in bulk solution; (B), when bound to the bead (grey circle). Fig. 8 – (A) Cysteines on FokI, where dyes are bound on the catalytic and DNA binding domains (B) FRET changes following the mixing of FokI (dye on catalytic domain) with DNA tagged with dye at either downstream end or upstream end. X-ray Crystallography Method – Crystal trays are set up using Phoenix robot and standard condition trays Rationale – To provide high resolution structures Enzyme – BcgI, with Nick Burton and Leo Brady (University of Bristol) Enzymes – SfiI, FokI with Aneel Agarwaal (New York) Analytical Ultracentrifugation (AUC) Method – Using AUC, the true MW of enzymes in solution and in association with DNA can be determined Rationale – Yields information about enzyme oligomerisation DNA in shape independent manner Enzymes tested – BcgI, FokI, SfiI, BspMI Method – Protein sample is nanosprayed into QTOF mass spectrometer Rationale – Provides MW values of species without fully disrupting non-covalent complexes Enzyme – BcgI Collaborators – Dr. Frank Sobott (University of Oxford) See poster from Marshall et al. - The Assembly of a restriction enzyme that cuts both strands on both sides of the site Native Mass Spec Fluorescence Resonance Energy Transfer (FRET) Fig. 10 – QTOF spectrum of BcgI with rapid desolvation to disrupt non-covalent complexes. AB complex confirms A-B interaction and excludes A-A interactions Bead Assay Method – 32 P-labelled oligos with a single recognition site are attached at low density to streptavidin beads Rationale – Reactions only occur at isolated sites as the oligos on the beads are too distant from each other for reactions in trans Enzymes tested – BcgI, SfiI, FokI, BspMI See Poster from Sanders et al - Targeting individual subunits of FokI restriction endonuclease to specific DNA strands Method – Fluorophores with overlapping spectra are placed on the enzyme and on the DNA (or on sites elsewhere in the protein), to give a FRET signal. Rationale – Reveals changes in the distance between the dyes upon DNA binding and cleavage, and in protein and/or protein-DNA conformational changes. Enzyme – FokI, SfiI Single Molecule Studies Method – For Tethered Particle Motion (TPM), the DNA rests between a bead and a cover slip. The bead motion is then proportional to the length of the tether. Rationale – Reveals DNA looping Enzymes – FokI, SfiI Collaborators – Niels Laurens and Gijs Wuite (VU, Amsterdam) See Poster from Rusling et al. - Exploring DNA-looping dynamics by the FokI restriction endonuclease 6.856.906.957.007.057.10 0.0 0.5 -2.0 0.0 1.0 2.0 Absorbance at 230 nm Centrifugal radius (cm) Residuals 0.25 MW app = 172 kDa Fig. 6 – In a TPM assay, bead motion is proportional to the length of DNA and thus reveals DNA looping events that shorten its length. (B) * * * * * * * * (A) (B) Streptavidin beads SfiI SfiI recognition site Radioactively labelled oligoduplex 0123456 Time (s) Fluorescence change (%) 2 4 6 8 10 Downstream Upstream 176,001 (A 2 B) 39,162 (BcgIB) 71,439 (native BcgIA) 110,733 (A 1 B 1 ) 71,566 (denatured BcgIA) Hint of larger aggregates? Cys 511 Cys 49 (A)