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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• meganuclease-induced recombination has long been limited by the repertoire of natural meganucleases, and the major limitation of the current technology is the requirement for the prior introduction of a meganuclease cleavage site in the locus of interest.
• the engineering of redesigned meganucleases cleaving chosen targets is under intense investigation.
• Such proteins could be used to cleave genuine chromosomal sequences and open new perspectives for genome engineering in wide range of appli- cations.
• meganucleases could be used to knock-out endogenous genes or knock-in exogenous sequences in the chromosome. It can as well be used for gene correction, and in principle, for the correction of mutations linked with monogenic diseases.
• meganucleases are essentially represented by homing endonucleases (HEs), a family of endonucleases encoded by mobile genetic elements, whose function is to initiate DNA double-strand break (DSB)-induced recombination events in a process referred to as homing (Chevalier and Stoddard, Nucleic Acids Res., 2001, 29, 3757-74; Kostriken et al, Cell; 1983, 35, 167-74; Jacquier and Dujon, Cell, 1985, 41, 383-94).
• HEs homing endonucleases
• DSB DNA double-strand break
• domain or “core domain” is intended the “LAGLIDADG homing endonuclease core domain” which is the characteristic ⁇ i ⁇ i ⁇ 2 ot2 ⁇ 3 ⁇ 4 ⁇ 3 fold of the homing endonucleases of the LAGLIDADG family, corresponding to a sequence of about one hundred amino acid residues.
• Said domain comprises four beta-strands ( ⁇ i , ⁇ 2, ⁇ 3, ⁇ 4 ) folded in an antiparallel beta-sheet which interacts with one half of the DNA target.
• viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
• retroviruses include: avian leukosis- sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retro viridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
• the subject-matter of the present invention is also the use of at least one heterodimeric meganuclease/single-chain meganuclease derivative, one or two polynucleotide(s), preferably included in expression vector(s), as defined above, for the preparation of a medicament for preventing, improving or curing a genetic disease in an individual in need thereof, said medicament being administrated by any means to said individual.
• the subject-matter of the present invention is also a method for preventing, improving or curing a genetic disease in an individual in need thereof, said method comprising at least the step of administering to said individual a composition as defined above, by any means.
• said double-stranded cleavage is induced, either in toto by administration of said meganuclease to an individual, or ex v/vo by introduction of said meganuclease into somatic cells removed from an individual and returned into the individual after modification.
• the subject-matter of the present invention is also the use of at least one heterodimeric meganuclease/single-chain meganuclease derivative, one or two polynucleotide(s), preferably included in expression vector(s), as defined above, in vitro, for inhibiting the propagation, inactivating or deleting an infectious agent that presents a DNA intermediate, in biological derived products or products intended for biological uses or for disinfecting an object.
• said composition comprises a targeting DNA construct comprising the sequence which repairs the site of interest flanked by sequences sharing homologies with the targeted locus as defined above.
• said targeting DNA construct is either included in a recombinant vector or it is included in an expression vector comprising the polynucleotide(s) encoding the meganuclease, as defined in the present invention.
• the heterodimeric meganuclease/single-chain meganuclease derivative may be advantageously associated with: liposomes, polyethyl- eneimine (PEI), and/or membrane translocating peptides (Bonetta, The Engineer, 2002, 16, 38; Ford et ah, Gene Ther., 2001, 8, 1-4 ; Wadia and Dowdy, Curr. Opin. Biotechnol., 2002, 13, 52-56); in the latter case, the sequence of the meganuclease fused with the sequence of a membrane translocating peptide (fusion protein).
• PEI polyethyl- eneimine
• Vectors comprising targeting DNA and/or nucleic acid encoding a meganuclease can be introduced into a cell by a variety of methods (e.g., injection, direct uptake, projectile bombardment, liposomes, electroporation). Meganucleases can be stably or transiently expressed into cells using expression vectors. Techniques of expression in eukaryotic cells are well known to those in the art. (See Current Protocols in Human Genetics: Chapter 12 "Vectors For Gene Therapy” & Chapter 13 "Delivery Systems for Gene Therapy”). Optionally, it may be preferable to incorporate a nuclear localization signal into the recombinant protein to be sure that it is expressed within the nucleus.
• heterodimeric meganuclease/single-chain meganuclease derivative and the methods of using said meganucleases according to the present invention include also the use of the polynucleotide(s), vector(s), cell, transgenic plant or non-human transgenic mammal encoding said heterodimeric meganuclease, as defined above.
• said heterodimeric meganuclease/single- chain meganuclease derivative, polynucleotide(s), vector(s), cell, transgenic plant or non-human transgenic mammal are associated with a targeting DNA construct as defined above.
• said vector encoding the monomer(s) of the heterodimeric meganuclease/single-chain meganuclease derivative comprises the targeting DNA construct, as defined above.
• the heterodimeric meganuclease according to the invention is derived from the monomers of the parent homodimeric LAGLIDADG endonucleases, according to standard site-directed mutagenesis methods which are well-known in the art and commercially available. They may be advantageously produced by amplifying overlapping fragments comprising each of the mutated positions, as defined above, according to well-known overlapping PCR techniques.
• SEQ ID NO: 3 and SEQ ID NO: 4 SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7or SEQ ID NO: 35 and SEQ ID NO: 8 may be used for amplifying the coding sequence (CDS) of monomer A and the three pairs SEQ ID NO: 9 and SEQ ID NO:10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 or SEQ ID NO: 36 and SEQ ID NO: 14 may be used for amplifying the CDS of monomer B.
• CDS coding sequence
• the two pairs SEQ ID NO: 37 and SEQ ID NO: 41, SEQ ID NO: 40 and SEQ ID NO: 42 may be used for amplifying the CDS of monomer A and the two pairs SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 may be used for amplifying the CDS of monomer B.
• the monomers of the parent homodimeric LAGLIDADG endonucleases may be obtained by a method for engineering variants able to cleave a genomic DNA target sequence of interest, as described previously in Smith et al,
• step (c) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant l-Cre ⁇ site wherein (i) the nucleotide triplet in positions -10 to -8 of the l-Crel site has been replaced with the nucleotide triplet which is present in positions -10 to -8 of said genomic target and (ii) the nucleotide triplet in positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions -10 to -8 of said genomic target,
• step (e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-Crel site wherein (i) the nucleotide triplet in positions +8 to +10 of the ⁇ -Cre ⁇ site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (ii) the nucleotide triplet in positions -10 to -8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +8 to +10 of said genomic target,
• step (f) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-Cre ⁇ site wherein (i) the nucleotide triplet in positions +3 to +5 of the l-Crel site has been replaced with the nucleotide triplet which is present in positions +3 to +5 of said genomic target and (ii) the nucleotide triplet in positions -5 to -3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (g) combining in a single variant, the mutation(s) in positions 26 to
• nucleotide triplet in positions -10 to -8 is identical to the nucleotide triplet which is present in positions -10 to -8 of said genomic target
• nucleotide triplet in positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions -10 to -8 of said genomic target
• nucleotide triplet in positions -5 to -3 is identical to the nucleotide triplet which is present in positions -5 to -3 of said genomic target
• nucleotide triplet in positions +3 to +5 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions -5 to -3 of said genomic target
• step (h) combining in a single variant, the mutation(s) in positions 26 to 40 and 44 to 77 of two variants from step (e) and step (f), to obtain a novel homodimeric l-Crel variant which cleaves a sequence wherein (i) the nucleotide triplet in positions +3 to +5 is identical to the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (ii) the nucleotide triplet in positions -5 to - 3 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (iii) the nucleotide triplet in positions +8 to +10 of the 1-OeI site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (iv) the nucleotide triplet in positions -10 to -8 is identical to the reverse complementary sequence of the nucleotide triplet in positions +8
• step (j) selecting and/or screening the heterodimers from step (i) which are able to cleave said genomic DNA target situated in a mammalian gene.
• Steps (a) and (b) may comprise the introduction of additional muta- tions in order to improve the binding and/or cleavage properties of the mutants, particularly at other positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target.
• steps may be performed by generating combinatorial libraries as described in the International PCT Application WO 2004/067736 and Arnould et al. (J. MoI. Biol., 2006, 355, 443-458).
• the selection and/or screening in steps (c), (d), (e), (f) and/or Q) may be performed by using a cleavage assay in vitro or in vivo, as described in the International PCT Application WO 2004/067736, Epinat et al.
• steps (c), (d), (e), (f) and/or (j) are performed in vivo, under conditions where the double-strand break in the mutated DNA target sequence which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break, as described in the International PCT Application WO 2004/067736, Epinat et al. (Nucleic Acids Res., 2003, 31, 2952-2962), Chames et al. (Nucleic Acids Res., 2005, 33, el 78), and Arnould et al. (J. MoI. Biol., 2006, 355, 443-458).
• the (intramolecular) combination of mutations in steps (g) and (h) may be performed by amplifying overlapping fragments comprising each of the two subdomains, according to well-known overlapping PCR techniques.
• step (g) and/or (h) may further comprise the introduction of random mutations on the whole variant or in a part of the variant, in particular the
• C-terminal half of the variant (positions 80 to 163). This may be performed by generating random mutagenesis libraries on a pool of variants, according to standard mutagenesis methods which are well-known in the art and commercially available.
• the (intermolecular) combination of the variants in step (i) is performed by co-expressing one variant from step (g) with one variant from step (h), so as to allow the formation of heterodimers.
• host cells may be modified by one or two recombinant expression vector(s) encoding said variant(s). The cells are then cultured under conditions allowing the expression of the variant(s), so that heterodimers are formed in the host cells, as described previously in the International PCT Application WO 2006/097854 and Arnould et al. (J. MoI. Biol., 2006, 355, 443-
• the mutations of monomers A as defined in the present invention are introduced in one monomer of the heterodimer obtained in step Q) and the mutations of monomer B as defined in the present invention, are introduced in the other monomer of said heterodimer.
• heterodimeric meganuclease of the invention may be obtained by a method derived from the hereabove method of engineering meganuclease variants, by introducing the following modifications:
• step (a) and step (b) are performed on two types of initial scaffold proteins: a first l-Crel scaffold having the mutations of monomer A and a second I- Cre ⁇ scaffold having the mutations of monomer B, and - the selection/screening of steps (c) to (f) is performed by transforming the library of variants having the mutations of monomer A or B as defined above in a host cell that expresses a 1-OeI mutant having the corresponding mutations (from monomer B or A, respectively) to allow the formation of heterodimers and selecting the functional heteodimeric variants by using a non- palindromic DNA target wherein one half of the l-Crel site is modified in positions ⁇ 3 to 5 or ⁇ 8 to 10 and the other half is not modified.
• steps (g) and (h) are performed by combining in a single variant, the mutations of two variants derived from the same monomer (A) or (B).
• Step (i) is performed by combining the variants derived from one of the monomers (A or B), obtained in step (g) with the variants derived from the other monomer, obtained in step (h) to form heterodimers.
• Single-chain meganucleases able to cleave a DNA target from the gene of interest are derived from the heterodimeric meganuclease according to the invention by methods well-known in the art (Epinat et al. 3 Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al., MoI. Cell., 2002, 10, 895-905; Steuer et al., Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). Any of such methods, may be applied for constructing the single- chain meganuclease of the invention.
• the polynucleotide sequence(s) encoding the monomers A and B as defined in the present invention may be prepared by any method known by the man skilled in the art. For example, they are amplified from a cDNA template, by polymerase chain reaction with specific primers. Preferably the codons of said cDNA are chosen to favour the expression of said protein in the desired expression system.
• the recombinant vector comprising said polynucleotides may be obtained and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques.
• the obligate heterodimer meganuclease of the invention is produced by co-expressing the monomers A and B as defined above, in a host cell or a transgenic animal/plant modified modified by one or two expression vector(s), under conditions suitable for the co-expression of the monomers, and the heterodimeric meganuclease is recovered from the host cell culture or from the transgenic animal/plant, by any appropriate means.
• the single-chain meganuclease of the invention is produced by expressing a fusion protein comprising the monomers A and B as defined above, in a host cell or a transgenic animal/plant modified modified by one expression vector, under conditions suitable for the expression of said fusion protein, and the single- chain meganuclease is recovered from the host cell culture or from the transgenic animal/plant, by any appropriate means.
• the subject-matter of the present invention is also the use of at least one heterodimeric meganuclease/single-chain meganuclease derivative, as defined above, as a scaffold for making other meganucleases.
• a third round of mutagenesis and selection/screening can be performed on the monomers, for the purpose of making novel, third generation homing endonucleases.
• the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the obligate heterodimer meganuclease according to the invention, as well as to the appended drawings in which:
• FIG. 1 represents a side view of the structure of the complex of meganuclease l-Cre- ⁇ (PDB:1G9Y) with its target DNA template.
• A A
• B B
• C Details of three modifiable interaction patches between the two monomers on the homodimer.
• D Designed heterodimeric interfaces, showing amino acid changes at each relevant position in the protein.
• FIG. 2 illustrates the effect of high salt concentration on the cleavage specificity.
• A QAN or
• B KTG protein was incubated with either the QAN homodimer site (GTT/AAC), the KTG homodimer DNA site (CCT/AGG), or a hybrid site QAN/KTG site (GTT/AGG), varying the concentration of NaCl between 50 and 300 mM.
• Arrows indicate the the uncut target DNA (3.2 kb) or the two bands resulting from digestion (1.1 and 2.1 kb).
• An asterisk (*) marks control lanes with DNA alone. 1 kb and lOObp ladders (FERMENTAS) are marked by M and Ml, respectively.
• FIG. 3 illustrates the CDS of the mutants derived from the I-Crel variants QAN and KTG designed for the making of the obligate heterodimer meganucleases.
• the sequences of the three mutagenizing oligos are underlined.
• QAN- Al and KTG-Al mutants oligos SEQ ID NO: 3, 35 and 5 respectively;
• QAN-A2 and KTG-A2 mutants oligos SEQ ID NO: 3, 7 and 5 respectively;
• QAN-B3 and KTG-B3 mutants oligos SEQ ID NO: 9, 11 and 13 respectively;
• QAN-B4 and KTG-B4 mutants oligos SEQ ID NO: 9, 11 and 36 respectively.
• FIG. 4 illustrates the expression and purification of the designed meganucleases.
• the target protein bands are marked by arrows.
• (A) Wild-type homodimers and mutant monomers. Lanes: M Standard Broad range markers (BIORAD); 1. Purified QANwt; 2. Purified KTGwt; 3. Pellet QAN-Al; 4. Supernatant QAN-Al; 5. Purified QAN-Al; 6. Pellet KTG-B3; 7. Supernatant KTG- B3; 8. Purified KTG-B3; 9. Pellet KTG-A2; 10. Supernatant KTG- A2; 11. Purified KTG-A2; 12. Pellet QAN-B3; 13. Supernatant QAN-B3; 14.
• FIG. 5 illustrates non-specific DNA cleavage and non-cleavage by singly-expressed designed meganuclease monomer variants under different salt conditions.
• Approximately 3.75 ⁇ M of each purified protein was incubated with 34 nM of purified plasmid (pre-linearized with Xmn ⁇ ), containing either the QAN homodimer site (GTT/AAC), the KTG homodimer DNA site (CCT/AGG), or a hybrid site QAN/KTG site (Q-K: GTT/AGG).
• the concentration of NaCl was either (A) 50 mM or (B) 225 mM.
• Arrows indicate the uncut target DNA (3.2 kb) or the two bands resulting from digestion (1.1 and 2.1 kb).
• An asterisk (*) marks control lanes with DNA alone. 1 kb ladders (FERMENTAS) are marked by M.
• FIG. 6 illustrates analytical ultracentrifugation of the different meganucleases.
• A The wild-type monomers form homodimers of about 400 kDa (KTG-wt; QAN-wt).
• B The designed non-homodimerising KTG-A2 and QAN-B3 form aggregates when expressed individually.
• C The co-expressed KTG-A2 and QAN-B3 form a perfect heterodimer.
• D The co-expressed QAN-B4 and KTG-A2 also form a heterodimer, to an extent, while QAN-Al and KTG-B3 do not.
• FIG. 7 illustrates specific DNA cleavage by co-expressed designed obligate heterodimer KTG-A2-QAN-B3 meganucleases.
• Approximately 3.75 ⁇ M of each purified protein was incubated with 34 nM of purified plasmid (pre- linearized with Xmn ⁇ ), containing either the QAN homodimer site (GTT/AAC), the KTG homodimer DNA site (CCT/AGG), or a hybrid site QAN/KTG site (Q-K: GTT/AGG).
• NaCl concentration was at 225 mM.
• Arrows indicate the uncut target DNA (3.2 kb) or the two bands resulting from digestion (1.1 and 2.1 kb). 1 kb ladders (FERMENTAS) are marked by M.
• - Figure 8 represents a bottom view of the structure of the complex of meganuclease l-Cre-l bound to DNA (PDB:1G9Y).
• A View of the complex showing the target DNA template.
• B The same view is represented but the DNA has been omitted.
• the two residues R51 and D 137 are represented in sticks and hydrogen bonds are represented by dashed lines.
• the circle in dashed lines delimits the active site, where the two DNA strands are cleaved.
• - Figure 9 illustrates the cleavage of RAG 1.10 by heterodimeric combinatorial mutants.
• the figure displays secondary screening of combinations of mutants of RAGl.10.2 and RAGl.10.3 cutters with the RAGl.10, RAGl.10.2 and RAGl.10.3 targets.
• the experiment format is an 2 by 2 dots format. The two dots forming the left column are the mutants and the right column is a control used to assess the quality of the experiment.
• l-Scel against an I-Scel target is in a and d, a low activity form of ⁇ -Sce ⁇ is in b and the empty vector is in c.
• Example 1 Protein Design
• the different heterodimers were designed using FoIdX (version 2.6.4), an automatic protein design algorithm (Guerois et al, J. MoI. Biol., 2002, 320,
• the second patch was chosen with the same idea of creating small electrostatic imbalances for homodimers, relative to heterodimers, but is positioned on each side of the coiled-coil; a double cluster of charged residues is made by the Lys96 and the Glu ⁇ l of each monomer (Figure IB).
• the second site was mutated with two arginines in monomer A, and two glutamates in monomer B, thus making a charged triangle in each monomer (positive in A, negative in B).
• the third region of interest is around the middle of the two helices involved in the interaction surface and is mainly composed of hydrophobic interactions and hydrogen bonds, making a kind of minicore.
• H-bond network is quite intricate and extends all the way to the active site, it was decided to perturb only the hydrophobic patch made by residues Tyrl2, Phel ⁇ , Val45, Trp53, Phe54, Leu55 and Leu58 of one monomer, with residue Leu97 of the other monomer (the latter acting like a cap closing the hydrophobic pocket) (Figure 1C).
• These two pockets were redesigned in order to introduce strong Van der Waals Clashes in the homodimers without disturbing the hydrophobic interactions in the heterodimers (i.e. without creating cavities and steric clashes).
• B4 differs from B3 by a mutation in Glycine at position 97 to accommodate with the Trp mutation of monomer A2 ( Figure ID).
• B5 and B6 differ from B3 and B4, respectively, by a substitution of Lys57 with a methionine, whereas Leu58 is not mutated.
• A2:B4 and A2_B6 presented a small decrease in interaction energy compared to the wild-type homodimer but was nonetheless significantly higher than the homodimers. Conversely, A1 :A1, A2:A2,
• B3:B3, B4:B4, B5:B5 and B6:B6 homodimers were all strongly destabilised and thus these species were expected to remain monomelic.
• Example 2 Engineering of meganucleases with different cleavage specificity and optimization of cleavage conditions
• Fresh BL21(DE3) (STRATAGENE) transformants carrying the pET (NOVAGEN) 1-Cre ⁇ mutants were grown overnight in 5 ml of Luria Broth (LB plus 30 ⁇ g/ml kanamycin) at 37 0 C on a shaker. This pre-culture was expanded to a larger culture (1 :200). At an OD 60O of 0.6-0.8, flasks were put on ice for 15 min to arrest growth. Expression was induced by adding IPTG (1 mM final) for 18 hours at 16°C, and cells were harvested by centrifugation (15 min, 16,000 g).
• Pellets were resuspended in 30 ml ice-cold lysis buffer (5OmM Tris-HCl pH 8, 20OmM NaCl, 5mM MgCl 2 , 10% Glycerol, 10 mM imidazole) containing 1 unit/ ⁇ l DNAse I and the procedure was carried out at 4 0 C thereafter. The suspension was immediately frozen in liquid nitrogen and thawed for 16 hours at 4 0 C on a rotating platform (60 rpm).
• 30 ice-cold lysis buffer 5OmM Tris-HCl pH 8, 20OmM NaCl, 5mM MgCl 2 , 10% Glycerol, 10 mM imidazole
• the suspension was homogenized with an ULTRA TURRAX T25 (JANKEL & KUNKEL, IKA-Labortechnik; 3 cycles of 1 min on ice) and then broken with an EmulsiFlex-C5 homogenizer (AVESTIN), for 5 rounds of 500-1000 psi (pounds per square inch) each.
• the lysate was centrifuged at 150,000 g for 60 min. This supernatant was cleared through a 0.45 ⁇ m filter (MILLIPORE).
• a 5ml Hi-Trap column (AMERSHAM-PHARMACIA) was loaded with 2 bead volumes (vol) of 250 mM NiSO 4 , and rinsed with 3 volumes of binding buffer (5OmM Tris-HCl pH 8, 30OmM NaCl, ImM DTT, 20% glycerol, 1OmM imidazole). The supernatant was then applied to the column and washed with washing buffer (binding buffer with 50 mM imidazole) until the A 2 so nm returned to its basal level. Protein was eluted with elution buffer (0.3M imidazole).
• the purified protein was aliquoted and snap-frozen in liquid nitrogen and stored at -8O 0 C. c) DNA digestion assays.
• the reaction mixture was prepared using 3.75 ⁇ M enzyme, 34 nM of purified 3.2 kb plasmid containing the appropriate target sequences (pre-linearized with Xmril ) and NaCl concentrations varying between 50 and 300 mM, in a 20 ⁇ l final reaction volume.
• the target DNA sequences are denoted by a 6-base code, with the first 3 bases corresponding to positions -5, -4 and -3 of the target sequence and the second 3 to positions +3, +4 and +5 on the same DNA strand; the two triplets are separated by a slash (/).
• the target of the KTG enzyme is cct/agg, that for the QAN target is gtt/aac, and the mixed DNA target for the heterodimer QAN-KTG is denominated as gtt/agg.
• KTG and QAN were each mutated at up to 6 amino acid positions to form two compatible heterodimerising interfaces, denoted KTG- A2 and QAN-B3. Mutations were introduced using round-the-world PCR with a Quickchange® kit (STRATAGENE, # 200518).
• KTG- A2 mutations (K7R, E8R, F54W, E61R, K96R, L97F) were introduced using three complementary primer sets:
• Al RR F (SEQ ID NO: 3) and A1_RR_R (SEQ ID NO:4): 5' caa tac caa ata taa cag gcg gtt cct get gta cct ggc eg 3' (SEQ ID NO: 3) 5'cgg cca ggt aca gca gga ace gcc tgt tat att tgg tat tg 3'(SEQ ID NO: 4); (ii) A1_RF_F (SEQ ID NO: 5) and A1_RF_R (SEQ ID NO: 6): 5 ' tea act gca gcc gtt tct gag att caa aca gaa aca ggc aaa cc 3 ' (SEQ ID NO: 5) 5' ggt ttg ct gttct gtt tga ate tea ga
• 5' cca gcg ccg ttg gtg get gga caa act agt gga tag aat tgg cgt tgg tta eg 3' (SEQ ID NO: 7)
• 5'cgt aac caa cgc caa ttc tat cca eta gtt tgt cca gcc ace aac ggc get gg 3 '(SEQ ID NO: 8).
• B3_EE_F (SEQ ID NO: 9) and B3JEEJR (SEQ ID NO: 10): 5' caa tac caa ata taa cga aga gtt cct get gta cct ggc eg 3' (SEQ ID NO: 9), and 5' egg cca ggt aca gca gga act ctt cgt tat att tgg tat tg 3' (SEQ ID NO: 10); (ii) B3_GME_F (SEQ ID NO: 11) and B3_GME_R (SEQ ID NO: 12): 5' cca gcg ccg ttg ggg tct gga caa aat ggt gga tga aat tgg cgt tgg tta eg 3' (SEQ ID NO: 11) 5 ' cgt aac ca
• the first primer set was used for PCR and transformation, according to the manufacturer's instructions (STRATAGENE, Quikchange®). Approximately 300 transformant bacterial colonies were pooled in 2 ml medium, and plasmid DNA was recovered by miniprep. This DNA was used as template for a second and then a third round of PCR with mutagenic primers. Five third-round mutants were verified by DNA sequencing.
• primers above are universal for any l-Crel mutant with altered specificity since the dimer interface mutations are outside the DNA recognition region.
• DNA digestion buffer consisting of: 25 mM HEPES (pH 8), 5 % Glycerol, 10 mM MgCl 2 , and 50 mM NaCl (low ionic strength) or 225 mM NaCl (high ionic strength). d) Analytical Centrifugation
• the oligomeric state of meganucl eases and mutants was investigated by monitoring sedimentation properties in centrifugation experiments; 1.04 mg of pure protein was used per sample (0.52 mg/ml of each monomer or 1.04 mg/ml of individual WT homodimers) in storage buffer (50 mM Tris-HCl pH 8.0, 225 raM
• the sedimentation velocity profiles were collected by monitoring the absorbance signal at 280 nm as the samples were centrifuged in a BECKMAN Optima XL-A centrifuge fitted with a four-hole AN-60 rotor and double-sector aluminium centerpieces (48 000 rpm, 4 °C).
• Molecular weight distributions were determined by the C(s) method implemented in the Sedfit (Schuck, P., Biophys., 2000, 78, 1606- 1619) and UltraScan 7.1 software packages (Demeler.B.,2005, http://www.ultrascan.uthscsa.edu )
• the designed mutants Al, A2, B3 and B4 were obtained by site- directed mutagenesis (STRATAGENE, QuikChange® Kit) of the original KTG and QAN enzyme expression vectors, followed by expression and purification of the corresponding proteins ( Figure 4).
• the QAN-Al, KTG-B3, KTG- A2, QAN-B3 and QAN-B4 mutants were selected These were designed to give coverage of all the designed heterodimer interactions A1:B3, A2:B3 and A2:B4, resulting in the heterodimers QAN-Al :KTG-B3, KTG-A2.-QAN- B3 and KTG-A2:QAN-B4.
• the double transformants were selected by growing the transformed colonies in presence of Kanamycin and Streptomycin-
• the RAGl.10.2 cutter with the sequence KRSNQS/AYSDR (residues at positions 28, 30, 32, 33, 38, 40 / 44, 68, 70, 75, 77 are indicated) and one RAGl.10.3 cutter with the sequence NNSSRR/YRSQV, previously obtained as described in Smith et al, Nucleic Acids Res., Epub 27 November 2006, and for each mutant, either the R51D mutation or the D137R mutation were introduced in order to create four single mutants.
• each mutation was introduced using two PCR reactions carried on the DNA of the RAGl.10.2 and RAGl.10.3 cutters.
• the first PCR reaction was performed with the primers: GaIlOF 5'-gcaactttagtgctgacacatacagg-3' (SEQ ID NO: 37) and R51DRev 5'-tttgtccagaaaccaacggtcctgggtcttttgagtcac-3' (SEQ ID NO: 38) and the second with the primers R51DFor 5'-gtgactcaaaagacccaggaccgttggttttctggacaac-3' (SEQ ID NO: 39) and GaIlOR 5'-acaaccttgattggagacttgacc-3' (SEQ ID NO: 40).
• Mating was performed using a colony gridder (QpixII, Genetix). Mutants were gridded on nylon filters covering YPD plates, using a low gridding density (about 4 spots/cm 2 ). A second gridding process was performed on the same filters to spot a second layer consisting of different reporter-harbouring yeast strains for each target. Membranes were placed on solid agar YPD rich medium, and incubated at 3O 0 C for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, adding G418, with galactose (1 %) as a carbon source, and incubated for five days at 37°C, to select for diploids carrying the expression and target vectors.

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