Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

1. Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands Kelly L. Sanders, Lucy E. Catto, Stuart R.W. Bellamy and Stephen E. Halford The DNA-Protein Interactions Unit, Department of Biochemistry, University of Bristol July 2009

2. 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’ 9 BASES 13 BASES Type IIS REases • recognise asymmetric DNA sequences and therefore cannot be recognised symmetrically by a homodimer • cleave both DNA strands at fixed positions downstream from recognition site Archetypal Type IIS: FokI • Previous data on FokI • Monomer in solution and when bound to DNA • Two domains: N-terminal DNA-binding domain – covers entire 5 bp recognition sequence C-terminal catalytic domain – single active site, with one PD…ExK motif These domains are joined by a flexible linker Cleavage of both strands achieved by dimerisation Recognition sequence - yellow box. DNA-binding domain – large blue oval Catalytic domain – small blue circle

3. D450A N13Y 1º monomer 2º monomer 1º monomer 2º monomer Crystal structure of FokI monomer bound to DNA Assembly of FokIDimer DNA Binding Domain EITHER 1° monomer at recognition site cuts top strand: 2o monomer cuts bottom stand. Cleavage Domain OR 1° monomer at recognition site cuts bottom strand: 2o monomer cuts top stand. +9 Cleavage sites +13 D450A – Asp450 forms part of the PD…EXK motif at the active site. Substitution with alanine abolishes catalytic activity without affecting dimerization or DNA recognition. N13Y – Substitution of Asn13 in the DNA recognition domain with a Tyr blocks sequence-specific binding, whilst retaining a functional cleavage/ dimerisation domain

4. Experimental strategy The catalytic domain of the 1o monomer could in principle engage the top strand 9 nts downstream of the site (pathway t): in this case, the 2o monomer attacks the bottom strand. Alternatively, the 1o monomer could attack the bottom strand 13 nts away (pathway b), whilst the 2o monomer attacks the top strand. Does FokI cleave both strands of DNA in sequential or parallel reactions? (A) With two monomers of wt FokI, both pathways (t) and (b) lead to cutting both strands. If reactions on both strands follow exponential progress curves starting from zero time, the 2 active sites must operate simultaneously – in parallel with each other. If FokI acts sequentially, cutting one particular strand before the other, then the product from cleaving the second strand will be formed only after an initial lag phase. (A) Wild-type/Wild-type (t) (b) Functional domains are shown in blue Domains inactivated by mutation are shown in red Which subunit cuts which strand? (B) With wt FokI and the D450A+N13Y double mutant, only the wt enzyme can bind to the recognition site and act as the 1º monomer so, if it acts by pathway (t), the top strand should be cleaved preferentially; if pathway (b) occurs, the bottom strand. (C) With the catalytically-defective D450A mutant and the binding-defective N13Y mutant, only D450A can bind to the recognition site, and N13Y has to function as the 2o monomer. But only N13Y is catalytically active. Consequently, pathway (t) should lead exclusively to bottom strand cleavage by the N13Y protein; pathway (b) to top strand cleavage. (B) Wild-type/N13Y-D450A (C) D450A/N13Y (t) (t) (b) (b)

5. 5’–BIO-CTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGG –3’ 3’-GACCGCTTTCCCCCTACACGACGTTCCGCTAATTCAACCTTG –5’ Experimental design • How to measure the reaction rate of an enzyme bound to one site when it can potentially bind two sites? - Need to eliminate trans reactions ? CHANGE • This can be achieved by immobilising the DNA on streptavidin-coated beads at a low density, to hold the DNA molecules separate from each other DNA substrate Klenow + 32P-dATP + dCTP Kinase + 32P-ATP. (Then Klenow + cold dATP + dCTP to fill in top) • 3 nt recession at the 3' end of the top strand was filled in using Klenow polymerase with dATP and dCTP to give a blunt-ended duplex 42 bp long, BIO-42. • Duplex radiolabelled in either top or bottom strand. - Top strand: dATP in the Klenow reaction was replaced with 32P[dATP]- Bottom strand: the 42 nt oligonucleotide was phosphorylated at its 5' end by polynucleotide kinase and γ32P[ATP]

6. 1 0.8 0.6 Fraction intact DNA Bottom 0.4 Top 0.2 0 5 nM wt FokI + 1 nM Bio-42 labelled in top or bottom strands 0 4 8 12 16 20 Time (min) TOP STRAND Time (min) BOTTOM STRAND Time (min) 0 20 0 20 S S P16 P12 Does FokI cleave both strands of DNA in sequential or parallel reactions? Sequential (consecutive) pathway OR Parallel pathway AND k = 0.3 min-1 k = 0.4 min-1 • Rate constant for cleavage of top strand equalled that of the bottom strand – active sites in both subunits of the dimer are equally active • Both reactions follow exponential progress curves – two strands cut in parallel reactions, rather than sequentially

7. Wild-type/N13Y-D450A: (t) (b) D450A/N13Y: (t) (b) Top strand Bottom strand 1 Time (min) Time (min) 0.8 M M 20 10 15 20 10 15 0 0 3 3 Top 1 2 4 5 1 2 4 5 0 1 0 1 0.6 S Fraction intact DNA 0.4 0.2 Bottom 0 4 8 12 16 20 0 0 4 8 12 16 20 P Time (min) Which subunit cuts which strand? 1 Bottom 0.8 S 0.6 Fraction intact DNA Top 0.4 0.2 P 0 Time (min) D450A can take up the 1o position but only N13Y has an intact catalytic domain, so whatever DNA cleavage is observed with this mixture can only come from N13Y as the 2o monomer. If the 2o monomer is limited to cutting one particular strand of the DNA, then this combination of mutants will cut only the strand attacked by the 2o monomer; the top strand The double mutant competes with the wt enzyme for the position of 2o monomer and so inhibits cleavage of the strand attacked by the 2o; the top strand • The bottom strand is cut by the 1o monomer and the top strand is cut by the 2o monomer • Combination of the N13Y and D450A variants cut DNA exclusively in the top strand – potential as sequence-specific strand–specific nicking reagent

8. A sequence-specific, strand-specific nicking endonuclease (NEase) from FokI • Only 8 NEases commercially available that cut DNA in a sequence-specific and strand-specific manner • Can be used to generate nicked or gapped duplexes for studies of DNA mismatch repair and for diagnostics • Numerous applications: - in strand displacement amplification - the detection and labelling of specific DNA sequences BbvCI • Majority constructed from heterodimeric enzymes - each subunit targets an individual strand in a non-palindromic sequence e.g BbvCI Nt.BbvCI Nb.BbvCI • Constructed here for a homodimeric enzyme – FokI - by using combinations of N13Y and D450A mutations

9. (A) D450A protein (B) N13Y protein 10nM D450A + N13Y 10nM N13Y + D450A 100 100 100 100 Wt 200 200 NE NE Wt 50 50 20 20 10 10 OC OC OC OC LIN LIN SC SC SC SC 200 200 NE NE Wt Wt 50 50 20 20 10 10 nicking observed > 50nM no detectable activity extent of nicking increased SC → OC Specificity of nicking • Needs to display minimal activity against the non-cognate strand and against non- cognate DNA sequences • D450A and N13Y variants tested against a 3kb plasmid with 1 FokI recognition site • Varied concentrations of each protein incubated with DNA (i) alone and (ii) in the presence of fixed conc. of the other protein Combination of the N13Y and D450A proteins can be used as a strand-specific, sequence specific nicking reagent * * limited to a relatively narrow range (<50nM) of N13Y

10. Summary • Wt FokI cuts the two DNA strands in parallel reactions - The first cut can occur in either the top or bottom strand with equal probability • The parallel reactions on the two strands can be assigned to individual subunits - 1º monomer at the recognition site cuts the bottom strand 13 nt downstream - 2º monomer cuts the top strand 9 nt downstream • Use of mutants excludes the possibility that either 1° or 2° monomers could switch their active sites between the strands • Two procedures were developed to direct the nuclease activity of FokI to a specific DNA strand - Addition of N13Y-D450A protein to a reaction of wt FokI inhibited cleavage of top strand - Combination of N13Y and D450A mutants exclusively cleaves top strand • The mixture of the N13Y and the D450A proteins have potential as a sequence-specific strand-specific nicking reagent- Applications in gene targeting - However, use of this system limited to a relatively narrow range of [N13Y]