% This file was created with JabRef 2.3.1. % Encoding: UTF8 @ARTICLE{Sasnauskas2010, author = {Giedrius Sasnauskas and Linas Zakrys and Mindaugas Zaremba and Richard Cosstick and James W Gaynor and Stephen E Halford and Virginijus Siksnys}, title = {A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3'-5' and 5'-3' strands by rotating a single active site.}, journal = {Nucleic Acids Res}, year = {2010}, volume = {38}, pages = {2399--2410}, number = {7}, month = {Apr}, abstract = {Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.}, doi = {10.1093/nar/gkp1194}, institution = {Institute of Biotechnology, LT-02241 Vilnius, Lithuania.}, keywords = {Amino Acid Substitution; Catalytic Domain; DNA; DNA Cleavage; Deoxyribonucleases, Type II Site-Specific; Dimerization; Histidine; Phosphates}, owner = {saulius}, pii = {gkp1194}, pmid = {20047964}, timestamp = {2010.10.13}, url = {http://dx.doi.org/10.1093/nar/gkp1194} } @ARTICLE{Zaremba2010, author = {Mindaugas Zaremba and Amelia Owsicka and Gintautas Tamulaitis and Giedrius Sasnauskas and Luda S Shlyakhtenko and Alexander Y Lushnikov and Yuri L Lyubchenko and Niels Laurens and Bram van den Broek and Gijs J L Wuite and Virginijus Siksnys}, title = {DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme.}, journal = {Nucleic Acids Res}, year = {2010}, month = {Jun}, abstract = {To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer-dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.}, doi = {10.1093/nar/gkq560}, institution = {Institute of Biotechnology, Graiciuno 8, LT-02241, Vilnius, Lithuania, Department of Pharmaceutical Sciences, College of Pharmacy, COP 1012, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA and Department of Physics and Astronomy and Laser Centre, VU University, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.}, owner = {saulius}, pii = {gkq560}, pmid = {20571089}, timestamp = {2010.10.13}, url = {http://dx.doi.org/10.1093/nar/gkq560} } @ARTICLE{Zaremba2010a, author = {Mindaugas Zaremba and Virginijus Siksnys}, title = {Molecular scissors under light control.}, journal = {Proc Natl Acad Sci U S A}, year = {2010}, volume = {107}, pages = {1259--1260}, number = {4}, month = {Jan}, doi = {10.1073/pnas.0913923107}, institution = {, Graiciuno 8, Vilnius LT-02241, Lithuania.}, keywords = {Animals; Azo Compounds; Biocatalysis; DNA Restriction Enzymes; Enzyme Activation; Humans; Light; Ultraviolet Rays}, owner = {saulius}, pii = {0913923107}, pmid = {20133886}, timestamp = {2010.10.13}, url = {http://dx.doi.org/10.1073/pnas.0913923107} } @ARTICLE{Gilmore2009, author = {Jamie L Gilmore and Yuki Suzuki and Gintautas Tamulaitis and Virginijus Siksnys and Kunio Takeyasu and Yuri L Lyubchenko}, title = {Single-molecule dynamics of the DNA-EcoRII protein complexes revealed with high-speed atomic force microscopy.}, journal = {Biochemistry}, year = {2009}, volume = {48}, pages = {10492--10498}, number = {44}, month = {Nov}, abstract = {The study of interactions of protein with DNA is important for gaining a fundamental understanding of how numerous biological processes occur, including recombination, transcription, repair, etc. In this study, we use the EcoRII restriction enzyme, which employs a three-site binding mechanism to catalyze cleavage of a single recognition site. Using high-speed atomic force microscopy (HS-AFM) to image single-molecule interactions in real time, we were able to observe binding, translocation, and dissociation mechanisms of the EcoRII protein. The results show that the protein can translocate along DNA to search for the specific binding site. Also, once specifically bound at a single site, the protein is capable of translocating along the DNA to locate the second specific binding site. Furthermore, two alternative modes of dissociation of the EcoRII protein from the loop structure were observed, which result in the protein stably bound as monomers to two sites or bound to a single site as a dimer. From these observations, we propose a model in which this pathway is involved in the formation and dynamics of a catalytically active three-site complex.}, doi = {10.1021/bi9010368}, institution = {Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025, USA.}, keywords = {DNA; Deoxyribonucleases, Type II Site-Specific; Microscopy, Atomic Force; Protein Binding}, owner = {saulius}, pmid = {19788335}, timestamp = {2010.10.13}, url = {http://dx.doi.org/10.1021/bi9010368} } @ARTICLE{Golovenko2009, author = {Dmitrij Golovenko and Elena Manakova and Giedre Tamulaitiene and Saulius Grazulis and Virginijus Siksnys}, title = {Structural mechanisms for the 5'-CCWGG sequence recognition by the N- and C-terminal domains of EcoRII.}, journal = {Nucleic Acids Res}, year = {2009}, volume = {37}, pages = {6613--6624}, number = {19}, month = {Oct}, abstract = {EcoRII restriction endonuclease is specific for the 5'-CCWGG sequence (W stands for A or T); however, it shows no activity on a single recognition site. To activate cleavage it requires binding of an additional target site as an allosteric effector. EcoRII dimer consists of three structural units: a central catalytic core, made from two copies of the C-terminal domain (EcoRII-C), and two N-terminal effector DNA binding domains (EcoRII-N). Here, we report DNA-bound EcoRII-N and EcoRII-C structures, which show that EcoRII combines two radically different structural mechanisms to interact with the effector and substrate DNA. The catalytic EcoRII-C dimer flips out the central T:A base pair and makes symmetric interactions with the CC:GG half-sites. The EcoRII-N effector domain monomer binds to the target site asymmetrically in a single defined orientation which is determined by specific hydrogen bonding and van der Waals interactions with the central T:A pair in the major groove. The EcoRII-N mode of the target site recognition is shared by the large class of higher plant transcription factors of the B3 superfamily.}, doi = {10.1093/nar/gkp699}, institution = {Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania.}, keywords = {Base Pairing; Base Sequence; DNA; DNA Methylation; Deoxyribonucleases, Type II Site-Specific; Models, Molecular; Protein Structure, Tertiary}, owner = {saulius}, pii = {gkp699}, pmid = {19729506}, timestamp = {2010.10.13}, url = {http://dx.doi.org/10.1093/nar/gkp699} } @ARTICLE{Ibryashkina2009, author = {Elena M Ibryashkina and Giedrius Sasnauskas and Alexander S Solonin and Marina V Zakharova and Virginijus Siksnys}, title = {Oligomeric structure diversity within the GIY-YIG nuclease family.}, journal = {J Mol Biol}, year = {2009}, volume = {387}, pages = {10--16}, number = {1}, month = {Mar}, abstract = {The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5'-CCGC/GG-3' ("/" marks the cleavage position) and cuts it to generate 3'-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.}, doi = {10.1016/j.jmb.2009.01.048}, institution = {Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.}, keywords = {Base Sequence; Biopolymers; DNA; Deoxyribonucleases, Type II Site-Specific; Dimerization; Hydrolysis; Kinetics; Protein Conformation}, owner = {saulius}, pii = {S0022-2836(09)00098-9}, pmid = {19361436}, timestamp = {2010.10.13}, url = {http://dx.doi.org/10.1016/j.jmb.2009.01.048} } @ARTICLE{Neely2009, author = {Robert K Neely and Gintautas Tamulaitis and Kai Chen and Marta Kubala and Virginijus Siksnys and Anita C Jones}, title = {Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes.}, journal = {Nucleic Acids Res}, year = {2009}, volume = {37}, pages = {6859--6870}, number = {20}, month = {Nov}, abstract = {Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.}, doi = {10.1093/nar/gkp688}, institution = {Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium. robert.neely@chem.kuleuven.be}, keywords = {DNA; DNA Restriction Enzymes; Deoxyribonucleases, Type II Site-Specific; Fluorescence; Nucleotides; Spectrometry, Fluorescence}, owner = {saulius}, pii = {gkp688}, pmid = {19740769}, timestamp = {2010.10.13}, url = {http://dx.doi.org/10.1093/nar/gkp688} } @ARTICLE{Gasiunas2008, author = {Giedrius Gasiunas and Giedrius Sasnauskas and Gintautas Tamulaitis and Claus Urbanke and Dalia Razaniene and Virginijus Siksnys}, title = {Tetrameric restriction enzymes: expansion to the GIY-YIG nuclease family.}, journal = {Nucleic Acids Res}, year = {2008}, volume = {36}, pages = {938--949}, number = {3}, month = {Feb}, abstract = {The GIY-YIG nuclease domain was originally identified in homing endonucleases and enzymes involved in DNA repair and recombination. Many of the GIY-YIG family enzymes are functional as monomers. We show here that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recognizes the symmetric sequence 5'-CCGC/GG-3' ('/' indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies provided here demonstrate that the Cfr42I tetramer is catalytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the homotetrameric Type IIF restriction enzymes that belong to the distinct PD-(E/D)XK nuclease superfamily. Unlike the PD-(E/D)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal-ion cofactors, including Mg2+, Mn2+, Co2+, Zn2+, Ni2+, Cu2+ and Ca2+. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar structural arrangement and phenotypes displayed by restriction enzymes of the PD-(E/D)XK and GIY-YIG nuclease families point to the functional significance of tetramerization.}, doi = {10.1093/nar/gkm1090}, institution = {Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania.}, keywords = {Amino Acid Sequence; Binding Sites; Cations, Divalent; DNA; Deoxyribonucleases, Type II Site-Specific; Electrophoretic Mobility Shift Assay; Kinetics; Metals; Molecular Sequence Data; Sequence Alignment; Substrate Specificity}, owner = {saulius}, pii = {gkm1090}, pmid = {18086711}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1093/nar/gkm1090} } @ARTICLE{Sasnauskas2008, author = {Giedrius Sasnauskas and Bernard A Connolly and Stephen E Halford and Virginijus Siksnys}, title = {Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme.}, journal = {Nucleic Acids Res}, year = {2008}, volume = {36}, pages = {3969--3977}, number = {12}, month = {Jul}, abstract = {Restriction endonucleases catalyse DNA cleavage at specific sites. The BfiI endonuclease cuts DNA to give staggered ends with 1-nt 3'-extensions. We show here that BfiI can also fill in the staggered ends: while cleaving DNA, it can add a 2'-deoxynucleoside to the reaction product to yield directly a blunt-ended DNA. We propose that nucleoside incorporation proceeds through a two-step reaction, in which BfiI first cleaves the DNA to make a covalent enzyme-DNA intermediate and then resolves it by a nucleophilic attack of the 3'-hydroxyl group of the incoming nucleoside, to yield a transesterification product. We demonstrate that base pairing of the incoming nucleoside with the protruding DNA end serves as a template for the incorporation and governs the yield of the elongated product. The efficiency of the template-directed process has been exploited by using BfiI for the site-specific modification of DNA 5'-termini with an amino group using a 5'-amino-5'-deoxythymidine.}, doi = {10.1093/nar/gkn343}, institution = {Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania.}, keywords = {DNA; Deoxyribonucleases, Type II Site-Specific; Deoxyribonucleosides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Templates, Genetic}, owner = {saulius}, pii = {gkn343}, pmid = {18515343}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1093/nar/gkn343} } @ARTICLE{Sukackaite2008, author = {Rasa Sukackaite and Saulius Grazulis and Matthias Bochtler and Virginijus Siksnys}, title = {The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution.}, journal = {J Mol Biol}, year = {2008}, volume = {378}, pages = {1084--1093}, number = {5}, month = {May}, abstract = {Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5'-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-A resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5'-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.}, doi = {10.1016/j.jmb.2008.03.041}, institution = {Institute of Biotechnology, Graiciuno 8, 02241 Vilnius, Lithuania.}, keywords = {Base Sequence; Crystallography, X-Ray; DNA; DNA Mutational Analysis; Deoxyribonucleases, Type II Site-Specific; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Protein Conformation}, owner = {saulius}, pii = {S0022-2836(08)00361-6}, pmid = {18433771}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.jmb.2008.03.041} } @ARTICLE{Szczepanowski2008, author = {Roman H Szczepanowski and Michael A Carpenter and Honorata Czapinska and Mindaugas Zaremba and Gintautas Tamulaitis and Virginijus Siksnys and Ashok S Bhagwat and Matthias Bochtler}, title = {Central base pair flipping and discrimination by PspGI.}, journal = {Nucleic Acids Res}, year = {2008}, volume = {36}, pages = {6109--6117}, number = {19}, month = {Nov}, abstract = {PspGI is a representative of a group of restriction endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a structure of PspGI with target DNA at 1.7 A resolution. In this structure, the bases at the center of the recognition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn conformation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrinsically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can contribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair.}, doi = {10.1093/nar/gkn622}, institution = {International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.}, keywords = {Adenine; Amino Acid Sequence; Base Pairing; Catalytic Domain; Crystallization; DNA; DNA-Binding Proteins; Deoxyribonucleases, Type II Site-Specific; Dimerization; Models, Molecular; Molecular Sequence Data; Nucleotides; Thymine}, owner = {saulius}, pii = {gkn622}, pmid = {18829716}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1093/nar/gkn622} } @ARTICLE{Tamulaitiene2008, author = {Giedre Tamulaitiene and Virginijus Siksnys}, title = {NotI is not boring.}, journal = {Structure}, year = {2008}, volume = {16}, pages = {497--498}, number = {4}, month = {Apr}, doi = {10.1016/j.str.2008.03.003}, keywords = {Crystallography, X-Ray; DNA; Deoxyribonucleases, Type II Site-Specific; Substrate Specificity}, owner = {saulius}, pii = {S0969-2126(08)00104-4}, pmid = {18400171}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.str.2008.03.003} } @ARTICLE{Tamulaitis2008, author = {Gintautas Tamulaitis and Mindaugas Zaremba and Roman H Szczepanowski and Matthias Bochtler and Virginijus Siksnys}, title = {How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets.}, journal = {Nucleic Acids Res}, year = {2008}, volume = {36}, pages = {6101--6108}, number = {19}, month = {Nov}, abstract = {Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This 'double check' mechanism explains their extraordinary specificity for an A/T pair in the flipping position.}, doi = {10.1093/nar/gkn621}, institution = {Institute of Biotechnology, Graiciuno 8, LT-02241, Vilnius, Lithuania.}, keywords = {Amino Acid Substitution; Base Pairing; Binding Sites; Catalytic Domain; DNA; Deoxyribonucleases, Type II Site-Specific; Electrophoretic Mobility Shift Assay; Models, Molecular; Substrate Specificity}, owner = {saulius}, pii = {gkn621}, pmid = {18820295}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1093/nar/gkn621} } @ARTICLE{Kaus-Drobek2007, author = {Magdalena Kaus-Drobek and Honorata Czapinska and Monika Sokołowska and Gintautas Tamulaitis and Roman H Szczepanowski and Claus Urbanke and Virginijus Siksnys and Matthias Bochtler}, title = {Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically.}, journal = {Nucleic Acids Res}, year = {2007}, volume = {35}, pages = {2035--2046}, number = {6}, abstract = {Restriction endonuclease MvaI recognizes the sequence CC/WGG (W stands for A or T, '/' designates the cleavage site) and generates products with single nucleotide 5'-overhangs. The enzyme has been noted for its tolerance towards DNA modifications. Here, we report a biochemical characterization and crystal structures of MvaI in an apo-form and in a complex with target DNA at 1.5 A resolution. Our results show that MvaI is a monomer and recognizes its pseudosymmetric target sequence asymmetrically. The enzyme consists of two lobes. The catalytic lobe anchors the active site residues Glu36, Asp50, Glu55 and Lys57 and contacts the bases from the minor grove side. The recognition lobe mediates all major grove interactions with the bases. The enzyme in the crystal is bound to the strand with T at the center of the recognition sequence. The crystal structure with calcium ions and DNA mimics the prereactive state. MvaI shows structural similarities to BcnI, which cleaves the related sequence CC/SGG and to MutH enzyme, which is a component of the DNA repair machinery, and nicks one DNA strand instead of making a double-strand break.}, doi = {10.1093/nar/gkm064}, institution = {International Institute of Molecular and Cell Biology, Warsaw, Poland.}, keywords = {Base Sequence; Catalytic Domain; Chromatography, Gel; Crystallography, X-Ray; DNA; DNA Methylation; Deoxyribonucleases, Type II Site-Specific; Models, Molecular; Protein Binding; Substrate Specificity; Ultracentrifugation}, owner = {saulius}, pii = {gkm064}, pmid = {17344322}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1093/nar/gkm064} } @ARTICLE{Sasnauskas2007, author = {Giedrius Sasnauskas and Bernard A Connolly and Stephen E Halford and Virginijus Siksnys}, title = {Site-specific DNA transesterification catalyzed by a restriction enzyme.}, journal = {Proc Natl Acad Sci U S A}, year = {2007}, volume = {104}, pages = {2115--2120}, number = {7}, month = {Feb}, abstract = {Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5' terminus of the cleaved DNA. Under certain conditions, the terminal 3'-OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a two-step mechanism. We propose that BfiI first makes a covalent enzyme-DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively.}, doi = {10.1073/pnas.0608689104}, institution = {Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania.}, keywords = {Bacillus; Binding Sites; Catalysis; DNA; DNA Restriction Enzymes; Deoxyribonucleases, Type II Site-Specific; Esterification; Hydrolysis; Nucleic Acid Conformation; Phospholipase D}, owner = {saulius}, pii = {0608689104}, pmid = {17267608}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1073/pnas.0608689104} } @ARTICLE{Shlyakhtenko2007, author = {Luda S Shlyakhtenko and Jamie Gilmore and Alex Portillo and Gintautas Tamulaitis and Virginijus Siksnys and Yuri L Lyubchenko}, title = {Direct visualization of the EcoRII-DNA triple synaptic complex by atomic force microscopy.}, journal = {Biochemistry}, year = {2007}, volume = {46}, pages = {11128--11136}, number = {39}, month = {Oct}, abstract = {Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein-DNA complexes. This is a ubiquitous phenomenon which is critical in various genetic processes. Although such interactions typically occur between two sites, interactions among three specific DNA regions have been identified, and a corresponding model has been proposed. Atomic force microscopy was used to test this model for the EcoRII restriction enzyme and provide direct visualization and characterization of synaptic protein-DNA complexes involving three DNA binding sites. The complex appeared in the images as a two-loop structure, and the length measurements proved the site specificity of the protein in the complex. The protein volume measurements showed that an EcoRII dimer is the core of the three-site synaptosome. Other complexes were identified and analyzed. The protein volume data showed that the dimeric form of the protein is responsible for the formation of other types of synaptic complexes as well. The applications of these results to the mechanisms of the protein-DNA interactions are discussed.}, doi = {10.1021/bi701123u}, institution = {Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025, USA.}, keywords = {DNA; Deoxyribonucleases, Type II Site-Specific; Microscopy, Atomic Force; Models, Molecular; Protein Binding}, owner = {saulius}, pmid = {17845057}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1021/bi701123u} } @ARTICLE{Sokolowska2007, author = {M. Sokolowska and M. Kaus-Drobek and H. Czapinska and G. Tamulaitis and V. Siksnys and M. Bochtler}, title = {Restriction endonucleases that resemble a component of the bacterial DNA repair machinery.}, journal = {Cell Mol Life Sci}, year = {2007}, volume = {64}, pages = {2351--2357}, number = {18}, month = {Sep}, abstract = {It has long been known that most Type II restriction endonucleases share a conserved core fold and similar active-sites. The same core folding motif is also present in the MutH protein, a component of the bacterial DNA mismatch repair machinery. In contrast to most Type II restriction endonucleases, which assemble into functional dimers and catalyze double-strand breaks, MutH is a monomer and nicks hemimethylated DNA. Recent biochemical and crystallographic studies demonstrate that the restriction enzymes BcnI and MvaI share many additional features with MutH-like proteins, but not with most other restriction endonucleases. The structurally similar monomers all recognize approximately symmetric target sequences asymmetrically. Differential sensitivities to slight substrate asymmetries, which could be altered by protein engineering, determine whether the enzymes catalyze only single-strand nicks or double-strand breaks.}, doi = {10.1007/s00018-007-7124-9}, institution = {International Institute of Molecular and Cell Biology, Trojdena 4, 02-109, Warsaw, Poland.}, keywords = {Bacteria; DNA Repair; DNA Restriction Enzymes; DNA, Bacterial}, owner = {saulius}, pmid = {17568994}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1007/s00018-007-7124-9} } @ARTICLE{Sokolowska2007a, author = {Monika Sokolowska and Magdalena Kaus-Drobek and Honorata Czapinska and Gintautas Tamulaitis and Roman H Szczepanowski and Claus Urbanke and Virginijus Siksnys and Matthias Bochtler}, title = {Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric complex with target DNA.}, journal = {J Mol Biol}, year = {2007}, volume = {369}, pages = {722--734}, number = {3}, month = {Jun}, abstract = {Restriction endonuclease BcnI cleaves duplex DNA containing the sequence CC/SGG (S stands for C or G, / designates a cleavage position) to generate staggered products with single nucleotide 5'-overhangs. Here, we show that BcnI functions as a monomer that interacts with its target DNA in 1:1 molar ratio and report crystal structures of BcnI in the absence and in the presence of DNA. In the complex with DNA, BcnI makes specific contacts with all five bases of the target sequence and not just with a half-site, as the protomer of a typical dimeric restriction endonuclease. Our data are inconsistent with BcnI dimerization and suggest that the enzyme introduces double-strand breaks by sequentially nicking individual DNA strands, although this remains to be confirmed by kinetic experiments. BcnI is remotely similar to the DNA repair protein MutH and shares approximately 20\% sequence identity with the restriction endonuclease MvaI, which is specific for the related sequence CC/WGG (W stands for A or T). As expected, BcnI is structurally similar to MvaI and recognizes conserved bases in the target sequence similarly but not identically. BcnI has a unique machinery for the recognition of the central base-pair.}, doi = {10.1016/j.jmb.2007.03.018}, institution = {International Institute of Molecular and Cell Biology, Warsaw, Poland.}, keywords = {Amino Acid Sequence; Base Sequence; Binding Sites; Crystallization; Crystallography, X-Ray; DNA; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Deoxyribonucleases, Type II Site-Specific; Dimerization; Endodeoxyribonucleases; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Protein Conformation}, owner = {saulius}, pii = {S0022-2836(07)00340-3}, pmid = {17445830}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.jmb.2007.03.018} } @ARTICLE{Sukackaite2007, author = {Rasa Sukackaite and Arunas Lagunavicius and Kornelijus Stankevicius and Claus Urbanke and Ceslovas Venclovas and Virginijus Siksnys}, title = {Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family.}, journal = {Nucleic Acids Res}, year = {2007}, volume = {35}, pages = {2377--2389}, number = {7}, abstract = {Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5'-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3'-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.}, doi = {10.1093/nar/gkm164}, institution = {Institute of Biotechnology, Graiciūno 8, Vilnius, Lithuania.}, keywords = {Archaeal Proteins; Base Sequence; Catalytic Domain; DNA; Deoxyribonucleases, Type II Site-Specific; Dimerization; Holliday Junction Resolvases; Molecular Sequence Data; Polymerase Chain Reaction; Protein Structure, Tertiary; Substrate Specificity}, owner = {saulius}, pii = {gkm164}, pmid = {17392342}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1093/nar/gkm164} } @ARTICLE{Tamulaitis2007, author = {Gintautas Tamulaitis and Mindaugas Zaremba and Roman H Szczepanowski and Matthias Bochtler and Virginijus Siksnys}, title = {Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence.}, journal = {Nucleic Acids Res}, year = {2007}, volume = {35}, pages = {4792--4799}, number = {14}, abstract = {Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI-DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.}, doi = {10.1093/nar/gkm513}, institution = {Institute of Biotechnology, Graiciuno 8, LT-02241, Vilnius, Lithuania.}, keywords = {2-Aminopurine; Calcium; Deoxyribonucleases, Type II Site-Specific; Fluorescent Dyes; Models, Molecular; Mutation; Nucleotides; Oligonucleotide Probes; Protein Structure, Tertiary}, owner = {saulius}, pii = {gkm513}, pmid = {17617640}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1093/nar/gkm513} } @ARTICLE{Bochtler2006, author = {Matthias Bochtler and Roman H Szczepanowski and Gintautas Tamulaitis and Saulius Grazulis and Honorata Czapinska and Elena Manakova and Virginijus Siksnys}, title = {Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease.}, journal = {EMBO J}, year = {2006}, volume = {25}, pages = {2219--2229}, number = {10}, month = {May}, abstract = {Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5'-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5'-overhangs. Here, we report the crystal structure of the Ecl18kI-DNA complex at 1.7 A resolution and compare it with the known structure of the NgoMIV-DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson-Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.}, doi = {10.1038/sj.emboj.7601096}, institution = {International Institute of Molecular and Cell Biology, Warsaw, Poland. MBochtler@iimcb.gov.pl}, keywords = {Amino Acid Sequence; Base Sequence; Binding Sites; Crystallography, X-Ray; DNA; Deoxyribonucleases, Type II Site-Specific; Dimerization; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Nucleotides; Protein Structure, Tertiary; Substrate Specificity}, owner = {saulius}, pii = {7601096}, pmid = {16628220}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1038/sj.emboj.7601096} } @ARTICLE{Tamulaitiene2006, author = {Giedre Tamulaitiene and Arturas Jakubauskas and Claus Urbanke and Robert Huber and Saulius Grazulis and Virginijus Siksnys}, title = {The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture.}, journal = {Structure}, year = {2006}, volume = {14}, pages = {1389--1400}, number = {9}, month = {Sep}, abstract = {Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.}, doi = {10.1016/j.str.2006.07.002}, institution = {Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania.}, keywords = {Amino Acid Sequence; Base Sequence; Crystallography; DNA Primers; DNA Restriction Enzymes; Dimerization; Hydrolysis; Models, Molecular; Molecular Sequence Data; Protein Conformation; Sequence Homology, Amino Acid}, owner = {saulius}, pii = {S0969-2126(06)00304-2}, pmid = {16962970}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.str.2006.07.002} } @ARTICLE{Tamulaitis2006, author = {Gintautas Tamulaitis and Giedrius Sasnauskas and Merlind Mucke and Virginijus Siksnys}, title = {Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease.}, journal = {J Mol Biol}, year = {2006}, volume = {358}, pages = {406--419}, number = {2}, month = {Apr}, abstract = {According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.}, doi = {10.1016/j.jmb.2006.02.024}, institution = {Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania.}, keywords = {Allosteric Site; Archaea; Binding Sites; DNA; Deoxyribonucleases, Type II Site-Specific; Hydrolysis; Kinetics; Plasmids; Protein Binding}, owner = {saulius}, pii = {S0022-2836(06)00208-7}, pmid = {16529772}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.jmb.2006.02.024} } @ARTICLE{Tamulaitis2006a, author = {Gintautas Tamulaitis and Merlind Mucke and Virginijus Siksnys}, title = {Biochemical and mutational analysis of EcoRII functional domains reveals evolutionary links between restriction enzymes.}, journal = {FEBS Lett}, year = {2006}, volume = {580}, pages = {1665--1671}, number = {6}, month = {Mar}, abstract = {The archetypal Type IIE restriction endonuclease EcoRII is a dimer that has a modular structure. DNA binding studies indicate that the isolated C-terminal domain dimer has an interface that binds a single cognate DNA molecule whereas the N-terminal domain is a monomer that also binds a single copy of cognate DNA. Hence, the full-length EcoRII contains three putative DNA binding interfaces: one at the C-terminal domain dimer and two at each of the N-terminal domains. Mutational analysis indicates that the C-terminal domain shares conserved active site architecture and DNA binding elements with the tetrameric restriction enzyme NgoMIV. Data provided here suggest possible evolutionary relationships between different subfamilies of restriction enzymes.}, doi = {10.1016/j.febslet.2006.02.010}, institution = {Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania.}, keywords = {Amino Acid Motifs; Binding Sites; Catalytic Domain; Chromatography, Gel; Conserved Sequence; DNA; DNA Mutational Analysis; DNA Restriction Enzymes; Deoxyribonucleases, Type II Site-Specific; Evolution, Molecular; Molecular Sequence Data; Protein Structure, Tertiary}, owner = {saulius}, pii = {S0014-5793(06)00198-0}, pmid = {16497303}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.febslet.2006.02.010} } @ARTICLE{Zaremba2006, author = {Mindaugas Zaremba and Giedrius Sasnauskas and Claus Urbanke and Virginijus Siksnys}, title = {Allosteric communication network in the tetrameric restriction endonuclease Bse634I.}, journal = {J Mol Biol}, year = {2006}, volume = {363}, pages = {800--812}, number = {4}, month = {Nov}, abstract = {Restriction endonuclease Bse634I is a homotetramer arranged as a dimer of two primary dimers. Bse634I displays its maximum catalytic efficiency upon binding of two copies of cognate DNA, one per each primary dimer. The catalytic activity of Bse634I on a single DNA copy is down-regulated due to the cross-talking interactions between the primary dimers. The mechanism of signal propagation between the individual active sites of Bse634I remains unclear. To identify communication pathways involved in the catalytic activity regulation of Bse634I tetramer we mutated a selected set of amino acid residues at the dimer-dimer interface and analysed the oligomeric state and catalytic properties of the mutant proteins. We demonstrate that alanine replacement of N262 and V263 residues located in the loop at the tetramerisation interface did not inhibit tetramer assembly but dramatically altered the catalytic properties of Bse634I despite of the distal location from the active site. Kinetic analysis using cognate hairpin oligonucleotide and one and two-site plasmids as substrates allowed us to identify two types of communication signals propagated through the dimer-dimer interface in the Bse634I tetramer: the inhibitory, or "stopper" and the activating, or "sync" signal. We suggest that the interplay between the two signals determines the catalytic and regulatory properties of the Bse634I and mutant proteins.}, doi = {10.1016/j.jmb.2006.08.050}, institution = {Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania.}, keywords = {Allosteric Regulation; DNA; DNA Cleavage; DNA Restriction Enzymes; Enzyme Stability; Guanidine; Kinetics; Models, Molecular; Mutagenesis; Nucleic Acid Conformation; Plasmids; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary}, owner = {saulius}, pii = {S0022-2836(06)01089-8}, pmid = {16987525}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.jmb.2006.08.050} } @ARTICLE{Grazulis2005, author = {Saulius Grazulis and Elena Manakova and Manfred Roessle and Matthias Bochtler and Giedre Tamulaitiene and Robert Huber and Virginijus Siksnys}, title = {Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease.}, journal = {Proc Natl Acad Sci U S A}, year = {2005}, volume = {102}, pages = {15797--15802}, number = {44}, month = {Nov}, abstract = {Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-A resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of BfiI exhibits a beta-barrel-like structure very similar to the effector DNA-binding domain of the Mg(2+)-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling BfiI catalytic activity in the absence of a specific DNA sequence. A psi-blast search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment.}, doi = {10.1073/pnas.0507949102}, institution = {Laboratory of Protein-DNA Interaction, Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania. grazulis@ibt.lt}, keywords = {Bacterial Proteins; Binding Sites; Catalytic Domain; Crystallography, X-Ray; DNA-Binding Proteins; Deoxyribonucleases; Deoxyribonucleases, Type II Site-Specific; Feedback, Biochemical; Molecular Structure; Protein Conformation}, owner = {saulius}, pii = {0507949102}, pmid = {16247004}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1073/pnas.0507949102} } @ARTICLE{Zaremba2005, author = {M. Zaremba and G. Sasnauskas and C. Urbanke and V. Siksnys}, title = {Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations.}, journal = {J Mol Biol}, year = {2005}, volume = {348}, pages = {459--478}, number = {2}, month = {Apr}, abstract = {The Bse634I restriction endonuclease is a tetramer and belongs to the type IIF subtype of restriction enzymes. It requires two recognition sites for its optimal activity and cleaves plasmid DNA with two sites much faster than a single-site DNA. We show that disruption of the tetramerisation interface of Bse634I by site-directed mutagenesis converts the tetrameric enzyme into a dimer. Dimeric W228A mutant cleaves plasmid DNA containing one or two sites with the same efficiency as the tetramer cleaves the two-site plasmid. Hence, the catalytic activity of the Bse634I tetramer on a single-site DNA is down-regulated due to the cross-talking interactions between the individual dimers. The autoinhibition within the Bse634I tetramer is relieved by bridging two DNA copies into the synaptic complex that promotes fast and concerted cleavage at both sites. Cleavage analysis of the oligonucleotide attached to the solid support revealed that Bse634I is able to form catalytically competent synaptic complexes by bridging two molecules of the cognate DNA, cognate DNA-miscognate DNA and cognate DNA-product DNA. Taken together, our data demonstrate that a single W228A mutation converts a tetrameric type IIF restriction enzyme Bse634I into the orthodox dimeric type IIP restriction endonuclease. However, the stability of the dimer towards chemical denaturants, thermal inactivation and proteolytic degradation are compromised.}, doi = {10.1016/j.jmb.2005.02.037}, institution = {Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania.}, keywords = {Bacillus stearothermophilus; Chromatography, Gel; DNA; DNA Restriction Enzymes; Dimerization; Enzyme Stability; Models, Molecular; Mutation; Plasmids; Protein Denaturation; Protein Folding; Protein Structure, Quaternary; Temperature; Tryptophan; Ultracentrifugation}, owner = {saulius}, pii = {S0022-2836(05)00203-2}, pmid = {15811381}, timestamp = {2009.02.21}, url = {http://dx.doi.org/10.1016/j.jmb.2005.02.037} } @comment{jabref-meta: selector_publisher:} @comment{jabref-meta: selector_author:} @comment{jabref-meta: selector_journal:} @comment{jabref-meta: selector_keywords:}