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• W1988031022 abstract "The hyperthermophilic archaeon Desulfurococcus mobilis I-DmoI protein belongs to the family of proteins known as homing endonucleases (HEs). HEs are highly specific DNA-cleaving enzymes that recognize long stretches of DNA and are powerful tools for genome engineering. Because of its monomeric nature, I-DmoI is an ideal scaffold for generating mutant enzymes with novel DNA specificities, similarly reported for homodimeric HEs, but providing single chain endonucleases instead of dimers. However, this would require the use of a mesophilic variant cleaving its substrate at temperatures of 37 °C and below. We have generated mesophilic mutants of I-DmoI, using a single round of directed evolution that relies on a functional assay in yeast. The effect of mutations identified in the novel proteins has been investigated. These mutations are located distant to the DNA-binding site and cause changes in the size and polarity of buried residues, suggesting that they act by destabilizing the protein. Two of the novel proteins have been produced and analyzed in vitro. Their overall structures are similar to that of the parent protein, but they are destabilized against thermal and chemical denaturation. The temperature-dependent activity profiles for the mutants shifted toward lower temperatures with respect to the wild-type activity profile. However, the most destabilized mutant was not the most active at low temperatures, suggesting that other effects, like local structural distortions and/or changes in the protein dynamics, also influence their activity. These mesophilic I-DmoI mutants form the basis for generating new variants with tailored DNA specificities. The hyperthermophilic archaeon Desulfurococcus mobilis I-DmoI protein belongs to the family of proteins known as homing endonucleases (HEs). HEs are highly specific DNA-cleaving enzymes that recognize long stretches of DNA and are powerful tools for genome engineering. Because of its monomeric nature, I-DmoI is an ideal scaffold for generating mutant enzymes with novel DNA specificities, similarly reported for homodimeric HEs, but providing single chain endonucleases instead of dimers. However, this would require the use of a mesophilic variant cleaving its substrate at temperatures of 37 °C and below. We have generated mesophilic mutants of I-DmoI, using a single round of directed evolution that relies on a functional assay in yeast. The effect of mutations identified in the novel proteins has been investigated. These mutations are located distant to the DNA-binding site and cause changes in the size and polarity of buried residues, suggesting that they act by destabilizing the protein. Two of the novel proteins have been produced and analyzed in vitro. Their overall structures are similar to that of the parent protein, but they are destabilized against thermal and chemical denaturation. The temperature-dependent activity profiles for the mutants shifted toward lower temperatures with respect to the wild-type activity profile. However, the most destabilized mutant was not the most active at low temperatures, suggesting that other effects, like local structural distortions and/or changes in the protein dynamics, also influence their activity. These mesophilic I-DmoI mutants form the basis for generating new variants with tailored DNA specificities. Homing endonucleases (HE), 5The abbreviations used are: HEhoming endonucleaseGdnHClguanidine hydrochloridePBSphosphate-buffered salineX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside.5The abbreviations used are: HEhoming endonucleaseGdnHClguanidine hydrochloridePBSphosphate-buffered salineX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. also known as meganucleases, produce double strand breaks in DNA, which induce the transposition of mobile intervening sequences, either introns or inteins, containing the endonuclease open reading frame into cognate alleles that lack this sequences, in a process known as homing (1Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (391) Google Scholar). HEs are highly sequence-specific enzymes, with recognition sites 12-45 bp long, and therefore have a very low frequency of cleavage even in complex genomes. Thus, HEs are very powerful tools for manipulating genomes of mammalian cells and plants (2Rouet P. Smih F. Jasin M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6064-6068Crossref PubMed Scopus (447) Google Scholar, 3Choulika A. Perrin A. Dujon B. Nicolas J.F. C. R. Acad. Sci. III. 1994; 317: 1013-1019PubMed Google Scholar, 4Choulika A. Perrin A. Dujon B. Nicolas J.F. Mol. Cell. Biol. 1995; 15: 1968-1973Crossref PubMed Scopus (367) Google Scholar) because of this rare cutting property. homing endonuclease guanidine hydrochloride phosphate-buffered saline 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. homing endonuclease guanidine hydrochloride phosphate-buffered saline 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. Sequence homology has been used to classify HEs into four families, the largest having the conserved LAGLIDADG sequence motif (5Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (375) Google Scholar). Homing endonucleases with only one such motif, such as I-CreI (6Wang J. Kim H.H. Yuan X. Herrin D.L. Nucleic Acids Res. 1997; 25: 3767-3776Crossref PubMed Scopus (44) Google Scholar), function as homodimers. By contrast, larger HEs containing two motifs, such as I-SceI (7Jacquier A. Dujon B. Cell. 1985; 41: 383-394Abstract Full Text PDF PubMed Scopus (277) Google Scholar) or I-DmoI (8Dalgaard J.Z. Garrett R.A. Belfort M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5414-5417Crossref PubMed Scopus (81) Google Scholar), are single chain enzymes. The three-dimensional structures of several LAGLIDADG endonucleases have been solved (9Flick K.E. McHugh D. Heath J.D. Stephens K.M. Monnat Jr., R.J. Stoddard B.L. Protein Sci. 1997; 6: 2677-2680Crossref PubMed Scopus (17) Google Scholar, 10Silva G.H. Dalgaard J.Z. Belfort M. Van Roey P. J. Mol. Biol. 1999; 286: 1123-1136Crossref PubMed Scopus (86) Google Scholar, 11Jurica M.S. Monnat Jr., R.J. Stoddard B.L. Mol. Cell. 1998; 2: 469-476Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 12Poland B.W. Xu M.Q. Quiocho F.A. J. Biol. Chem. 2000; 275: 16408-16413Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Chevalier B.S. Monnat Jr., R.J. Stoddard B.L. Nat. Struct. Biol. 2001; 8: 312-316Crossref PubMed Scopus (95) Google Scholar, 14Duan X. Gimble F.S. Quiocho F.A. Cell. 1997; 89: 555-564Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 15Werner E. Wende W. Pingoud A. Heinemann U. Nucleic Acids Res. 2002; 30: 3962-3971Crossref PubMed Google Scholar, 16Ichiyanagi K. Ishino Y. Ariyoshi M. Komori K. Morikawa K. J. Mol. Biol. 2000; 300: 889-901Crossref PubMed Scopus (97) Google Scholar, 17Moure C.M. Gimble F.S. Quiocho F.A. J. Mol. Biol. 2003; 334: 685-695Crossref PubMed Scopus (84) Google Scholar, 18Spiegel P.C. Chevalier B. Sussman D. Turmel M. Lemieux C. Stoddard B.L. Structure (Lond.). 2006; 14: 869-880Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 19Nakayama H. Shimamura T. Imagawa T. Shirai N. Itoh T. Sako Y. Miyano M. Sakuraba H. Ohshima T. Nomura N. Tsuge H. J. Mol. Biol. 2006; 365: 362-378Crossref PubMed Scopus (10) Google Scholar). These proteins adopt a similar active conformation; the single chain LAGLIDADG proteins display two distinct domains with 2-fold pseudo-symmetry, similar to the perfect 2-fold symmetry of the homodimeric proteins. The two LAGLIDADG motifs form structurally conserved α-helices tightly packed at the center of the interdomain or intermonomer interface. On either side of the LAGLIDADG α-helices, a four-stranded β-sheet provides a DNA-binding interface that drives the base-specific interaction of the protein with each one of the half-sites of the target DNA sequence (11Jurica M.S. Monnat Jr., R.J. Stoddard B.L. Mol. Cell. 1998; 2: 469-476Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 13Chevalier B.S. Monnat Jr., R.J. Stoddard B.L. Nat. Struct. Biol. 2001; 8: 312-316Crossref PubMed Scopus (95) Google Scholar). The last acidic residue of the LAGLIDADG motif, located at the C-terminal end of the corresponding helix, participates in DNA cleavage by metal-dependent phosphodiester hydrolysis (13Chevalier B.S. Monnat Jr., R.J. Stoddard B.L. Nat. Struct. Biol. 2001; 8: 312-316Crossref PubMed Scopus (95) Google Scholar). Several hundred HEs have been identified to date, but the probability of finding an HE cleavage site in a chosen gene is still very low. Thus, generating artificial meganucleases with tailored specificity by rational design or mutagenesis and screening and/or selection is a field of intense research (20Ashworth J. Havranek J.J. Duarte C.M. Sussman D. Monnat Jr., R.J. Stoddard B.L. Baker D. Nature. 2006; 441: 656-659Crossref PubMed Scopus (271) Google Scholar, 21Gimble F.S. Moure C.M. Posey K.L. J. Mol. Biol. 2003; 334: 993-1008Crossref PubMed Scopus (57) Google Scholar, 22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar, 23Doyon J.B. Pattanayak V. Meyer C.B. Liu D.R. J. Am. Chem. Soc. 2006; 128: 2477-2484Crossref PubMed Scopus (101) Google Scholar, 24Steuer S. Pingoud V. Pingoud A. Wende W. ChemBioChem. 2004; 5: 206-213Crossref PubMed Scopus (36) Google Scholar, 25Seligman L.M. Chisholm K.M. Chevalier B.S. Chadsey M.S. Edwards S.T. Savage J.H. Veillet A.L. Nucleic Acids Res. 2002; 30: 3870-3879Crossref PubMed Scopus (96) Google Scholar). Recently, we used a semi-rational mutagenesis approach coupled with high throughput screening to derive hundreds of novel endonucleases from I-CreI, an HE with locally altered specificity from the LAGLIDADG family (22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar). We also used a combinatorial and rational approach for engineering the overall specificity of these proteins and for designing an endonuclease that cleaves sequences from the human RAG1 and XPC genes (26Smith J. Grizot S. Arnould S. Duclert A. Epinat J.C. Chames P. Prieto J. Redondo P. Blanco F.J. Bravo J. Montoya G. Paques F. Duchateau P. Nucleic Acids Res. 2006; 34: e149Crossref PubMed Scopus (238) Google Scholar, 27Arnould S. Perez C. Cabaniols J.P. Smith J. Gouble A. Grizot S. Epinat J.C. Duclert A. Duchateau P. Paques F. J. Mol. Biol. 2007; 371: 49-65Crossref PubMed Scopus (121) Google Scholar). However, these meganucleases are heterodimers, consisting of two engineered monomers that need to be co-expressed in the targeted cell, a characteristic stemming from the homodimeric nature of I-CreI. As a consequence, we actually produce two undesired homodimers in addition to the heterodimer. We know that natural and engineered meganucleases tolerate a certain degree of degeneracy (5Chevalier B.S. Stoddard B.L. Nucleic Acids Res. 2001; 29: 3757-3774Crossref PubMed Scopus (375) Google Scholar, 22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar, 27Arnould S. Perez C. Cabaniols J.P. Smith J. Gouble A. Grizot S. Epinat J.C. Duclert A. Duchateau P. Paques F. J. Mol. Biol. 2007; 371: 49-65Crossref PubMed Scopus (121) Google Scholar), and these molecules may contribute to a loss of specificity, as has been observed with zinc finger nucleases, another class of engineered endonucleases (28Bibikova M. Golic M. Golic K.G. Carroll D. Genetics. 2002; 161: 1169-1175Crossref PubMed Google Scholar). One possible way of bypassing this issue is to use single chain scaffolds. Previous studies have described the development of single chain I-CreI versions (29Epinat J.C. Arnould S. Chames P. Rochaix P. Desfontaines D. Puzin C. Patin A. Zanghellini A. Paques F. Lacroix E. Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (186) Google Scholar), or of hybrid-homing endonucleases, by fusing two LAGLIDADG nucleases, I-DmoI and I-CreI. DmoCre (29Epinat J.C. Arnould S. Chames P. Rochaix P. Desfontaines D. Puzin C. Patin A. Zanghellini A. Paques F. Lacroix E. Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (186) Google Scholar) and E-DreI (30Chevalier B.S. Kortemme T. Chadsey M.S. Baker D. Monnat R.J. Stoddard B.L. Mol. Cell. 2002; 10: 895-905Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) are two similar proteins, differing only in the linker region; both cleave novel hybrid DNA targets consisting of two moieties, one from the I-CreI cleavage site, the other from the I-DmoI cleavage site. However, both proteins have shown intrinsic limits: the single chain I-CreI molecule is imperfectly folded (29Epinat J.C. Arnould S. Chames P. Rochaix P. Desfontaines D. Puzin C. Patin A. Zanghellini A. Paques F. Lacroix E. Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (186) Google Scholar) and probably as a consequence is difficult to engineer 6S. Grizot, S. Arnould, P. Duchateau, and F. Pâques, unpublished results.6S. Grizot, S. Arnould, P. Duchateau, and F. Pâques, unpublished results.; and the I-DmoI/I-CreI chimeras have maintained I-DmoI thermophilic properties (29Epinat J.C. Arnould S. Chames P. Rochaix P. Desfontaines D. Puzin C. Patin A. Zanghellini A. Paques F. Lacroix E. Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (186) Google Scholar, 30Chevalier B.S. Kortemme T. Chadsey M.S. Baker D. Monnat R.J. Stoddard B.L. Mol. Cell. 2002; 10: 895-905Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Another limit of the I-CreI scaffold is the number of sequences that can be targeted by engineered derivatives. One of the most elusive factors in DNA binding and cleavage by I-CreI is the role of the four central nucleotides of the DNA target molecule. No direct contact has been reported between the protein residues and the DNA four central bases of the DNA; however, several nucleotide substitutions in this region result in a total loss of cleavage by I-CreI and its engineered derivatives (27Arnould S. Perez C. Cabaniols J.P. Smith J. Gouble A. Grizot S. Epinat J.C. Duclert A. Duchateau P. Paques F. J. Mol. Biol. 2007; 371: 49-65Crossref PubMed Scopus (121) Google Scholar, 31Argast G.M. Stephens K.M. Emond M.J. Monnat Jr., R.J. J. Mol. Biol. 1998; 280: 345-353Crossref PubMed Scopus (100) Google Scholar). Previous attempts at modifying the specificity of homing endonucleases were generally based on the prior identification of direct interaction patterns (21Gimble F.S. Moure C.M. Posey K.L. J. Mol. Biol. 2003; 334: 993-1008Crossref PubMed Scopus (57) Google Scholar, 22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar, 25Seligman L.M. Chisholm K.M. Chevalier B.S. Chadsey M.S. Edwards S.T. Savage J.H. Veillet A.L. Nucleic Acids Res. 2002; 30: 3870-3879Crossref PubMed Scopus (96) Google Scholar, 26Smith J. Grizot S. Arnould S. Duclert A. Epinat J.C. Chames P. Prieto J. Redondo P. Blanco F.J. Bravo J. Montoya G. Paques F. Duchateau P. Nucleic Acids Res. 2006; 34: e149Crossref PubMed Scopus (238) Google Scholar, 32Sussman D. Chadsey M. Fauce S. Engel A. Bruett A. Monnat Jr., R. Stoddard B.L. Seligman L.M. J. Mol. Biol. 2004; 342: 31-41Crossref PubMed Scopus (66) Google Scholar). However, given the absence of such information, it may be very difficult to change the recognition pattern in the central part of the I-CreI target. Again, using other scaffolds may address this issue, and they would ideally be monomeric (see above). I-DmoI is a well characterized HE, encoded by an archaeal intron from the hyperthermophile archaeon Desulfurococcus mobilis (8Dalgaard J.Z. Garrett R.A. Belfort M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5414-5417Crossref PubMed Scopus (81) Google Scholar, 10Silva G.H. Dalgaard J.Z. Belfort M. Van Roey P. J. Mol. Biol. 1999; 286: 1123-1136Crossref PubMed Scopus (86) Google Scholar, 33Lykke-Andersen J. Thi-Ngoc H.P. Garrett R.A. Nucleic Acids Res. 1994; 22: 4583-4590Crossref PubMed Scopus (29) Google Scholar, 34Silva G.H. Belfort M. Nucleic Acids Res. 2004; 32: 3156-3168Crossref PubMed Scopus (28) Google Scholar, 35Aagaard C. Awayez M.J. Garrett R.A. Nucleic Acids Res. 1997; 25: 1523-1530Crossref PubMed Scopus (35) Google Scholar). I-DmoI is an attractive scaffold for engineering novel DNA sequence specificities, as its monomeric nature allows for the generation of single chain mutants with altered specificities along the whole DNA recognition sequence. Screening for novel specificities in living cells requires that the activity is detectable at mesophilic temperatures. However, I-DmoI, as expected for an endonuclease from a hyperthermophilic organism, is essentially active at high temperatures, displaying no activity or residual activity at 37 °C (8Dalgaard J.Z. Garrett R.A. Belfort M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5414-5417Crossref PubMed Scopus (81) Google Scholar). In this study, we generated I-DmoI variants with enhanced cleavage activity at physiological temperatures in yeast. Two of the proteins were characterized in vitro and were less stable and more active at lower temperatures than the wild type. These novel I-DmoI variants could be used as initial scaffolds for engineering proteins that cleave new DNA targets, using a similar strategy to that described previously for I-CreI (22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar, 26Smith J. Grizot S. Arnould S. Duclert A. Epinat J.C. Chames P. Prieto J. Redondo P. Blanco F.J. Bravo J. Montoya G. Paques F. Duchateau P. Nucleic Acids Res. 2006; 34: e149Crossref PubMed Scopus (238) Google Scholar, 27Arnould S. Perez C. Cabaniols J.P. Smith J. Gouble A. Grizot S. Epinat J.C. Duclert A. Duchateau P. Paques F. J. Mol. Biol. 2007; 371: 49-65Crossref PubMed Scopus (121) Google Scholar). Construction of Mutant Libraries and Target Vectors–A library of I-DmoI variant open reading frames was generated as follows: the first 300 bp of the wild-type I-DmoI-encoding gene were amplified by mutagenic PCR (36Cadwell R.C. Joyce G.F. PCR Methods Applications. 1992; 2: 28-33Crossref PubMed Scopus (819) Google Scholar, 37Cadwell R.C. Joyce G.F. PCR Methods Applications. 1994; 3: S136-S140Crossref PubMed Scopus (314) Google Scholar), whereas the second half of the gene was amplified with a high fidelity enzyme. For mutagenic PCR, we used classic PCR conditions known to enhance the natural mutation rate of a Taq polymerase. MgCl2 concentration was increased to stabilize DNA mismatches, and MnCl2 was added to decrease the fidelity of the enzyme, and the concentrations of dCTP, dTTP, and polymerase were increased to favor misincorporation, as described in previous reports (36Cadwell R.C. Joyce G.F. PCR Methods Applications. 1992; 2: 28-33Crossref PubMed Scopus (819) Google Scholar, 37Cadwell R.C. Joyce G.F. PCR Methods Applications. 1994; 3: S136-S140Crossref PubMed Scopus (314) Google Scholar). Under these conditions, the mutation rate was measured to be 6.6 × 10-3 per nucleotide resulting in 96% of mutated molecules, each one with one to six mutations (36Cadwell R.C. Joyce G.F. PCR Methods Applications. 1992; 2: 28-33Crossref PubMed Scopus (819) Google Scholar). Both PCR amplifications overlapped. This allowed a second conservative amplification, reconstituting a full-length I-DmoI gene; the first part contained the mutated domain (from the ATG start codon to the beginning of the second LAGLIDADG domain), and the second part contained the wild-type encoding sequence (from the second LAGLIDADG domain to the stop codon). The resulting PCR products were introduced into the 2-μm-based replicative vector pCLS542 marked with the LEU2 gene and transformed into the Saccharomyces cerevisiae selection strain (see below) as described previously (22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar). Yeast reporter vectors were constructed as described previously (22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar). Construction of Target Clones–The natural I-DmoI DNA target sequence 5′-GCCTTGCCGGGTAAGTTCCGGCGC-3′ (38Dalgaard J.Z. Garrett R.A. Belfort M. J. Biol. Chem. 1994; 269: 28885-28892Abstract Full Text PDF PubMed Google Scholar) was cloned using the Gateway protocol (Invitrogen) into the yeast reporter vector pFL39-ADH-LACURAZ, which contains a control I-SceI target site, as described previously (21Gimble F.S. Moure C.M. Posey K.L. J. Mol. Biol. 2003; 334: 993-1008Crossref PubMed Scopus (57) Google Scholar, 22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar). The yeast reporter vector was transformed into the S. cerevisiae strain FYBL2-7B (MATa, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202). Selection in Yeast–A yeast strain, specific for selecting endonucleases that cleave the I-DmoI related targets, was constructed with strain FYC2-6A (MATα, trp1Δ63, leu2Δ1, his3Δ200), as described previously (39Chames P. Epinat J.C. Guillier S. Patin A. Lacroix E. Paques F. Nucleic Acids Res. 2005; 33: e178Crossref PubMed Scopus (53) Google Scholar). The LYS2 endogenous gene was disrupted using the “pop-in” transformation method (40Hicks J.B. Hinnen A. Fink G.R. Cold Spring Harbor Symp. Quant. Biol. 1979; 43: 1305-1313Crossref PubMed Google Scholar), resulting in two truncated but overlapping lys2 genes, separated by a cassette containing a kanamycin resistance gene and the I-DmoI target site. The ADE2 gene was similarly disrupted, resulting in two truncated ade2 copies separated by an I-DmoI site and a TRP1 gene (39Chames P. Epinat J.C. Guillier S. Patin A. Lacroix E. Paques F. Nucleic Acids Res. 2005; 33: e178Crossref PubMed Scopus (53) Google Scholar). Cleavage of the I-DmoI sites resulted in functional LYS2 and ADE2 genes by tandem repeat recombination (Fig. 1a). The final selection yeast strain was G418-resistant, Trp+, Ade-, and Lys-. For selection, yeast cells from the selection strain were transformed with the I-DmoI mutant library, marked with the LEU2 gene. Cells were plated onto a selective medium lacking leucine with glucose (2%) as a carbon source. After 3 days of growth at 37 °C, colonies were suspended in water, and aliquots of the suspension were plated onto selective media lacking leucine, adenine, and lysine, and containing galactose (2%). Leu+, Ade+, and Lys+ isolates were picked and rearrayed for screening. Screening in Yeast–After selection, Leu+, Ade+, and Lys+ isolates were screened for their ability to induce tandem repeat recombination in a LacZ reporter plasmid carrying an I-DmoI cleavage site. For this, isolates were mated with the FYBL2-7B (MATa, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202) yeast strain carrying the reporter system described in Fig. 1b, and diploids were grown at 37 °C on selective media containing galactose (2%). Meganuclease-induced recombination of the LacZ reporter system restores a functional β-galactosidase gene, which can be detected by X-gal staining. Mating, cleavage induction, and X-gal staining were performed as described previously (22Arnould S. Chames P. Perez C. Lacroix E. Duclert A. Epinat J.C. Stricher F. Petit A.S. Patin A. Guillier S. Rolland S. Prieto J. Blanco F.J. Bravo J. Montoya G. Serrano L. Duchateau P. Paques F. J. Mol. Biol. 2006; 355: 443-458Crossref PubMed Scopus (167) Google Scholar). Sequencing of Positive Clones–DNA was extracted from positive clones using Lyticase (from Sigma L2524); 1.5 ml of saturated cultures (microtubes or deep well plates) were centrifuged, and the pellet was resuspended with 50 μl of 33.5 mm KH2PO4, pH 7.5. Lyticase (20 μl of 2.5 units/μl) was added, and the samples were incubated at 37 °C for 1 h. Spheroplasts were lysed by hypo-osmotic shock by adding 10 μl of SDS (20% stock). The debris was removed by centrifugation. An aliquot of the supernatant was used to transform Escherichia coli and amplify plasmid DNA. Two E. coli isolates were selected for each yeast-positive isolate. Plasmid DNA was then extracted and sequenced. Protein Expression and Purification–For expression in E. coli, the I-DmoI gene was obtained as described (29Epinat J.C. Arnould S. Chames P. Rochaix P. Desfontaines D. Puzin C. Patin A. Zanghellini A. Paques F. Lacroix E. Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (186) Google Scholar), and the I-DmoI mutants, D1 and D2, were generated using the QuikChange XL site-directed mutagenesis kit (Stratagene). This construct is 200 residues long and differs from the native I-DmoI protein by an extra Ala residue at the N terminus and an AAALEHHHHHH sequence at the C-terminal end for affinity purification. This recombinant enzyme is active (29Epinat J.C. Arnould S. Chames P. Rochaix P. Desfontaines D. Puzin C. Patin A. Zanghellini A. Paques F. Lacroix E. Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (186) Google Scholar) and corresponds to the sequence coded by the linear form of the intron after excision; this linear form is 6 residues shorter at the C-terminal end than that coded by the circularized intron (8Dalgaard J.Z. Garrett R.A. Belfort M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5414-5417Crossref PubMed Scopus (81) Google Scholar). Both forms have very similar biochemical properties (38Dalgaard J.Z. Garrett R.A. Belfort M. J. Biol. Chem. 1994; 269: 28885-28892Abstract Full Text PDF PubMed Google Scholar). In the crystal structure of the short enzyme, the last 9 residues are not observed (41Dalgaard J.Z. Silva G.H. Belfort M. Van Roey P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1435-1436Crossref PubMed Google Scholar), indicating that the C-terminal end is flexible. The genes were inserted into pET-24d (+) vectors (Novagen) and overexpressed in E. coli Rosetta(DE3)pLysS at 25 °C. The protein purification protocol was similar to that described for I-CreI homing endonucleases (27Arnould S. Perez C. Cabaniols J.P. Smith J. Gouble A. Grizot S. Epinat J.C. Duclert A. Duchateau P. Paques F. J. Mol. Biol. 2007; 371: 49-65Crossref PubMed Scopus (121) Google Scholar, 42Prieto J. Redondo P. Padro D. Arnould S. Epinat J.C. Paques F. Blanco F.J. Montoya G. Nucleic Acids Res. 2007; 35: 3262-3271Crossref PubMed Scopus (21) Google Scholar). Pure proteins in phosphate-buffered saline (PBS, 137 mm NaCl, 10 mm sodium phosphate, 2.7 mm KCl, 2 mm potassium phosphate, pH 7.4) were flash-frozen with liquid nitrogen and stored at -80 °C until use. Overloaded Coomassie-stained SDS-polyacrylamide gels showed that the proteins were almost 100% pure. The identity of the proteins was confirmed by mass spectrometry, which indicated that the initial methionine was not present in the purified proteins. Protein concentrations were measured by ultraviolet absorbance using an extinction coefficient of 25,900 m-1 cm-1, calculated from the amino acid sequence using the ProtParam web server. Circular Dichroism (CD) Analysis–Far-UV CD spectra (250-190 nm) were recorded at 20 °C on a Jasco-810 dichrograph equipped with a Peltier thermoelectric temperature controller, previously calibrated with d-10-camphorsulfonic acid. The spectra were acquired in the continuous mode with 2 nm bandwidth, 4-s response, and a scan speed of 100 nm/min. We used 0.1-cm path length quartz cuvettes (Hellma) and protein samples with a concentration of 150 μm in PBS for CD analysis. Ten scans were accumulated to obtain the final spectra. Thermal denaturation curves were obtained at a protein concentration of 10 μm in a 2-mm cuvette. The ellipticity at 222 nm from 5 to 95 °C was recorded at 1 °C/min intervals. NMR Spectroscopy–1H NMR spectra were recorded at 25 °C in a Bruker AVANCE 600 spectrometer equipped with a cryoprobe. The concentrations of protein samples were 500 μm in PBS plus 5% 2H2O. 2,2-Dimethyl-2-silapentane-5-sulfonate sodium salt was used as internal proton chemical shift reference. Analytical Ultracentrifugation–Sedimentation velocity and equilibrium experiments were performed on 200 μm protein samples in PBS at 20 °C, as described previously for I-CreI proteins (42Prieto J. Redondo P. Padro D. Arnould S. Epinat J.C. Paques F. Blanco F.J. Montoya G. Nucleic Acids Res. 2007; 35: 3262-3271Crossref PubMed Scopus (21) Google Scholar). Chemical Denaturation–I-DmoI, D1, and D2 proteins were unfolded using guanidine hydrochloride (GdnHCl, Sigma). A stock solution of 8 m GdnHCl was gravimetrically prepared at 20 °C i" @default.
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• W1988031022 date "2008-02-01" @default.
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• W1988031022 title "Generation and Analysis of Mesophilic Variants of the Thermostable Archaeal I-DmoI Homing Endonuclease" @default.
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