The Experts below are selected from a list of 10950 Experts worldwide ranked by ideXlab platform
Alfred Pingoud - One of the best experts on this subject based on the ideXlab platform.
-
type ii Restriction Endonucleases a historical perspective and more
Nucleic Acids Research, 2014Co-Authors: Alfred Pingoud, Geoffrey G Wilson, Wolfgang WendeAbstract:This article continues the series of Surveys and Summaries on Restriction Endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures.
-
on the divalent metal ion dependence of dna cleavage by Restriction Endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Monika Fuxreiter, Peter Friedhoff, Monika Reuter, Letif Mones, Alfred PingoudAbstract:Abstract Restriction Endonucleases of the PD…D/EXK family need Mg2+ for DNA cleavage. Whereas Mg2+ (or Mn2+) promotes catalysis, Ca2+ (without Mg2+) only supports DNA binding. The role of Mg2+ in DNA cleavage by Restriction Endonucleases has elicited many hypotheses, differing mainly in the number of Mg2+ involved in catalysis. To address this problem, we measured the Mg2+ and Mn2+ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me2+ per active site. DNA cleavage experiments were carried out at various Mg2+ and Mn2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg2+ and Mn2+ concentration dependence. In general, the Mg2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg2+ or Mn2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca2+. Based on these results, we propose that one Mg2+ or Mn2+ is critical for Restriction enzyme activation, and binding of a second Me2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg2+ or Mn2+ mainly leads to an increase in Km, such that the inhibitory effect of excess Mg2+ or Mn2+ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me2+ binding to these enzymes.
-
on the divalent metal ion dependence of dna cleavage by Restriction Endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Monika Fuxreiter, Peter Friedhoff, Monika Reuter, Letif Mones, Alfred PingoudAbstract:Restriction Endonucleases of the PD...D/EXK family need Mg(2+) for DNA cleavage. Whereas Mg(2+) (or Mn(2+)) promotes catalysis, Ca(2+) (without Mg(2+)) only supports DNA binding. The role of Mg(2+) in DNA cleavage by Restriction Endonucleases has elicited many hypotheses, differing mainly in the number of Mg(2+) involved in catalysis. To address this problem, we measured the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me(2+) per active site. DNA cleavage experiments were carried out at various Mg(2+) and Mn(2+) concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg(2+) and Mn(2+) concentration dependence. In general, the Mg(2+) concentration optimum (between approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 0.1 and 1 mM). At still higher Mg(2+) or Mn(2+) concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca(2+). Based on these results, we propose that one Mg(2+) or Mn(2+) is critical for Restriction enzyme activation, and binding of a second Me(2+) plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg(2+) or Mn(2+) mainly leads to an increase in K(m), such that the inhibitory effect of excess Mg(2+) or Mn(2+) can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me(2+) binding to these enzymes.
-
type ii Restriction Endonucleases structure and mechanism
Cellular and Molecular Life Sciences, 2005Co-Authors: Alfred Pingoud, Monika Fuxreiter, Vera Pingoud, Wolfgang WendeAbstract:Type II Restriction Endonucleases are components of Restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few Restriction Endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II Restriction Endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II Restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.
-
a fluorimetric assay for on line detection of dna cleavage by Restriction Endonucleases
Journal of Biotechnology, 2002Co-Authors: Kristin Eisenschmidt, Albert Jeltsch, Thomas Lanio, Alfred PingoudAbstract:We have developed an assay for online detection of DNA cleavage by Restriction Endonucleases, suitable for the high throughput screening of the activity and flanking sequence preference of Restriction endonuclease variants. For this purpose oligodeoxynucleotides were used, labeled with either 6-FAM or TAMRA whose fluorescence is quenched by a neighboring DABCYL group. After endonucleolytic cleavage the products are too short to remain double-stranded and the fluorophor labeled strand is released with concomitant increase in fluorescence which can be easily quantified. Employing this method, cleavage reactions can be monitored continuously, allowing for fast detection of specific activity as well as determination of kinetic parameters. To demonstrate the reliability of our assay we measured KM and kcat values for the Restriction endonuclease EcoRV and obtained results similar to those obtained with established assays. Moreover, our method makes it possible to observe the cleavage of two different substrates differing in the sequences flanking the EcoRV site and labeled with different fluorophors in competition in a single experiment. This assay can be carried out in a microplate format, which allows for the analysis of many Restriction endonuclease variants in parallel.
Matthias Bochtler - One of the best experts on this subject based on the ideXlab platform.
-
protein domain guided screen for sequence specific and phosphorothioate dependent Restriction Endonucleases
Frontiers in Microbiology, 2020Co-Authors: Thomas Lutz, Daniel F Heiter, Honorata Czapinska, Alexey Fomenkov, Vladimir Potapov, Bo Cao, Peter C Dedon, Matthias BochtlerAbstract:Modification dependent Restriction Endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2-4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA. We report five SBD-HNH Endonucleases, all recognizing GpsAAC/GpsTTC sequence and cleaving outside with a single nucleotide 3' stagger: EcoWI (N7/N6), Ksp11411I (N5/N4), Bsp305I (N6/N4-5), Mae9806I [N(8-10)/N(8-9)], and Sau43800I [N(8-9)/N(7-8)]. EcoWI and Bsp305I are more specific for PT modified DNA in Mg2+ buffer, and promiscuous with Mn2+. Ksp11411I is more PT specific with Ni2+. EcoWI and Ksp11411I cleave fully- and hemi-PT modified oligos, while Bsp305I cleaves only fully modified ones. EcoWI forms a dimer in solution and cleaves more efficiently in the presence of two modified sites. In addition, we demonstrate that EcoWI PT-dependent activity has biological function: EcoWI expressing cells restrict dnd+ GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent Restriction Endonucleases (PTDRs).
-
Restriction Endonucleases that cleave rna dna heteroduplexes bind dsdna in a like conformation
Nucleic Acids Research, 2020Co-Authors: Honorata Czapinska, Marlena Kisiala, Monika Kowalska, Michal Pastor, Henryk J Korza, Matthias BochtlerAbstract:Restriction Endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of Restriction Endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA/DNA hybrid. The specific complex reveals a novel form of semi-specific dsDNA readout by a hexa-coordinated metal cation, most likely Ca2+ or Mg2+. Substitutions of residues anchoring this non-catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A-like form. This creates space for 2'-OH groups to be accommodated without intra-nucleic acid steric conflicts. PD-(D/E)XK Restriction Endonucleases of known structure that bind their dsDNA targets in the A-like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA/DNA heteroduplexes. We conclude that A-form dsDNA binding is a good predictor for RNA/DNA cleavage activity.
-
a protein architecture guided screen for modification dependent Restriction Endonucleases
Nucleic Acids Research, 2019Co-Authors: Thomas Lutz, Honorata Czapinska, Alexey Fomenkov, Kiersten Flodman, Alyssa Copelas, Megumu Mabuchi, Matthias BochtlerAbstract:Modification dependent Restriction Endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing Endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the Endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing Endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.
-
Restriction Endonucleases that resemble a component of the bacterial dna repair machinery
Cellular and Molecular Life Sciences, 2007Co-Authors: Virginijus Siksnys, Honorata Czapinska, Matthias Bochtler, M Sokolowska, Magdalena Kausdrobek, G TamulaitisAbstract: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.
Albert Jeltsch - One of the best experts on this subject based on the ideXlab platform.
-
on the divalent metal ion dependence of dna cleavage by Restriction Endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Monika Fuxreiter, Peter Friedhoff, Monika Reuter, Letif Mones, Alfred PingoudAbstract:Abstract Restriction Endonucleases of the PD…D/EXK family need Mg2+ for DNA cleavage. Whereas Mg2+ (or Mn2+) promotes catalysis, Ca2+ (without Mg2+) only supports DNA binding. The role of Mg2+ in DNA cleavage by Restriction Endonucleases has elicited many hypotheses, differing mainly in the number of Mg2+ involved in catalysis. To address this problem, we measured the Mg2+ and Mn2+ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me2+ per active site. DNA cleavage experiments were carried out at various Mg2+ and Mn2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg2+ and Mn2+ concentration dependence. In general, the Mg2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg2+ or Mn2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca2+. Based on these results, we propose that one Mg2+ or Mn2+ is critical for Restriction enzyme activation, and binding of a second Me2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg2+ or Mn2+ mainly leads to an increase in Km, such that the inhibitory effect of excess Mg2+ or Mn2+ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me2+ binding to these enzymes.
-
on the divalent metal ion dependence of dna cleavage by Restriction Endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Monika Fuxreiter, Peter Friedhoff, Monika Reuter, Letif Mones, Alfred PingoudAbstract:Restriction Endonucleases of the PD...D/EXK family need Mg(2+) for DNA cleavage. Whereas Mg(2+) (or Mn(2+)) promotes catalysis, Ca(2+) (without Mg(2+)) only supports DNA binding. The role of Mg(2+) in DNA cleavage by Restriction Endonucleases has elicited many hypotheses, differing mainly in the number of Mg(2+) involved in catalysis. To address this problem, we measured the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me(2+) per active site. DNA cleavage experiments were carried out at various Mg(2+) and Mn(2+) concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg(2+) and Mn(2+) concentration dependence. In general, the Mg(2+) concentration optimum (between approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 0.1 and 1 mM). At still higher Mg(2+) or Mn(2+) concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca(2+). Based on these results, we propose that one Mg(2+) or Mn(2+) is critical for Restriction enzyme activation, and binding of a second Me(2+) plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg(2+) or Mn(2+) mainly leads to an increase in K(m), such that the inhibitory effect of excess Mg(2+) or Mn(2+) can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me(2+) binding to these enzymes.
-
a fluorimetric assay for on line detection of dna cleavage by Restriction Endonucleases
Journal of Biotechnology, 2002Co-Authors: Kristin Eisenschmidt, Albert Jeltsch, Thomas Lanio, Alfred PingoudAbstract:We have developed an assay for online detection of DNA cleavage by Restriction Endonucleases, suitable for the high throughput screening of the activity and flanking sequence preference of Restriction endonuclease variants. For this purpose oligodeoxynucleotides were used, labeled with either 6-FAM or TAMRA whose fluorescence is quenched by a neighboring DABCYL group. After endonucleolytic cleavage the products are too short to remain double-stranded and the fluorophor labeled strand is released with concomitant increase in fluorescence which can be easily quantified. Employing this method, cleavage reactions can be monitored continuously, allowing for fast detection of specific activity as well as determination of kinetic parameters. To demonstrate the reliability of our assay we measured KM and kcat values for the Restriction endonuclease EcoRV and obtained results similar to those obtained with established assays. Moreover, our method makes it possible to observe the cleavage of two different substrates differing in the sequences flanking the EcoRV site and labeled with different fluorophors in competition in a single experiment. This assay can be carried out in a microplate format, which allows for the analysis of many Restriction endonuclease variants in parallel.
-
structure and function of type ii Restriction Endonucleases
Nucleic Acids Research, 2001Co-Authors: Alfred Pingoud, Albert JeltschAbstract:More than 3000 type II Restriction Endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4–8 bp and, in the presence of Mg 2+ , cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of Restriction enzymes show a common structural core comprising four β-strands and one α-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-lik ee nzymes). Like other DNA binding proteins, Restriction enzymes are capable of nonspecific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically ∼15–20 hydrogen bonds are formed between a dimeric Restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many Restriction enzymes the catalytic centers, one in each subunit, are represented by the PD ...D /EXK motif, in which the two carboxylates are responsible for Mg 2+ binding, the essential cofactor
-
recognition and cleavage of dna by type ii Restriction Endonucleases
FEBS Journal, 1997Co-Authors: Alfred Pingoud, Albert JeltschAbstract:Restriction Endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II Restriction Endonucleases are homodimers which recognize short palindromic sequences 4–8 bp in length and, in the presence of Mg2+, cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10–12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any Restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriciton Endonucleases are not only functionally but also evolutionarily related.
Michal Hocek - One of the best experts on this subject based on the ideXlab platform.
-
polymerase synthesis of dnas bearing vinyl groups in the major groove and their cleavage by Restriction Endonucleases
ChemBioChem, 2014Co-Authors: Michaela Mackova, Michal Hocek, Radek PohlAbstract:DNA molecules containing 5-vinyluracil, 5-vinylcytosine, or 7-deaza-7-vinyladenine were prepared by polymerase incorporation of the corresponding vinyl-modified 2′-deoxyribonucleoside triphosphates, and the influence of the vinyl group in the major groove of DNA on the cleavage by diverse type II Restriction Endonucleases (REs) was studied. The presence of 5-vinyluracil was tolerated by most of the REs, whereas only some REs were able to cleave sequences containing 7-deaza-7-vinyladenine. The enzyme ScaI was found to cleave DNA containing 5-vinylcytosine efficiently but not DNA containing the related 5-ethynylcytosine. All other REs failed to cleave sequences containing any cytosine modifications.
-
Polymerase Synthesis of Photocaged DNA Resistant against Cleavage by Restriction Endonucleases
Angewandte Chemie, 2014Co-Authors: Zuzana Vaníková, Michal HocekAbstract:5-[(2-Nitrobenzyl)oxymethyl]-2′-deoxyuridine 5′-O-triphosphate was used for polymerase (primer extension or PCR) synthesis of photocaged DNA that is resistant to the cleavage by Restriction Endonucleases. Photodeprotection of the caged DNA released 5-hydroxymethyluracil-modified nucleic acids, which were fully recognized and cleaved by Restriction enzymes.
-
cleavage of functionalized dna containing 5 modified pyrimidines by type ii Restriction Endonucleases
ChemBioChem, 2011Co-Authors: Hana Macickovacahova, Radek Pohl, Michal HocekAbstract:A series of six pyrimidine-modified dNTPs--5-ethynyl-, 5-phenyl-, and 5-(3-nitrophenyl)deoxycitidine and -deoxyuridine triphosphates--were prepared and incorporated by primer extension with Vent (exo-)polymerase to specific DNA sequences within or next to the recognition sequences of selected Restriction Endonucleases. The cleavage of these pyrimidine-modified DNA sequences by 13 Restriction enzymes was then studied. Whereas the presence of any modified C within the target sequence completely prevented any Restriction cleavage, most enzymes tolerated the presence of 5-ethynylU and two of them even the presence of 5-phenyl- and 5-(3-nitrophenyl)U. Modifications outside the recognition sequence were tolerated except in the case of phenyl derivatives with the PvuII enzyme. 5-EthynylC was used for protection of the recognition sequence from cleavage in the presence of the second unmodified copy of the same sequence that was cleaved.
-
cleavage of adenine modified functionalized dna by type ii Restriction Endonucleases
Nucleic Acids Research, 2009Co-Authors: Hana Macickovacahova, Michal HocekAbstract:A set of 6 base-modified 2′-deoxyadenosine derivatives was incorporated to diverse DNA sequences by primer extension using Vent (exo-) polymerase and the influence of the modification on cleavage by diverse Restriction Endonucleases was studied. While 8-substituted (Br or methyl) adenine derivatives were well tolerated by the Restriction enzymes and the corresponding sequences were cleaved, the presence of 7-substituted 7-deazaadenine in the recognition sequence resulted in blocking of cleavage by some enzymes depending on the nature and size of the 7-substituent. All sequences with modifications outside of the recognition sequence were perfectly cleaved by all the Restriction enzymes. The results are useful both for protection of some sequences from cleavage and for manipulation of functionalized DNA by Restriction cleavage.
Wolfgang Wende - One of the best experts on this subject based on the ideXlab platform.
-
type ii Restriction Endonucleases a historical perspective and more
Nucleic Acids Research, 2014Co-Authors: Alfred Pingoud, Geoffrey G Wilson, Wolfgang WendeAbstract:This article continues the series of Surveys and Summaries on Restriction Endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures.
-
on the divalent metal ion dependence of dna cleavage by Restriction Endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Monika Fuxreiter, Peter Friedhoff, Monika Reuter, Letif Mones, Alfred PingoudAbstract:Abstract Restriction Endonucleases of the PD…D/EXK family need Mg2+ for DNA cleavage. Whereas Mg2+ (or Mn2+) promotes catalysis, Ca2+ (without Mg2+) only supports DNA binding. The role of Mg2+ in DNA cleavage by Restriction Endonucleases has elicited many hypotheses, differing mainly in the number of Mg2+ involved in catalysis. To address this problem, we measured the Mg2+ and Mn2+ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me2+ per active site. DNA cleavage experiments were carried out at various Mg2+ and Mn2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg2+ and Mn2+ concentration dependence. In general, the Mg2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg2+ or Mn2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca2+. Based on these results, we propose that one Mg2+ or Mn2+ is critical for Restriction enzyme activation, and binding of a second Me2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg2+ or Mn2+ mainly leads to an increase in Km, such that the inhibitory effect of excess Mg2+ or Mn2+ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me2+ binding to these enzymes.
-
on the divalent metal ion dependence of dna cleavage by Restriction Endonucleases of the ecori family
Journal of Molecular Biology, 2009Co-Authors: Vera Pingoud, Albert Jeltsch, Jürgen Alves, Wolfgang Wende, Monika Fuxreiter, Peter Friedhoff, Monika Reuter, Letif Mones, Alfred PingoudAbstract:Restriction Endonucleases of the PD...D/EXK family need Mg(2+) for DNA cleavage. Whereas Mg(2+) (or Mn(2+)) promotes catalysis, Ca(2+) (without Mg(2+)) only supports DNA binding. The role of Mg(2+) in DNA cleavage by Restriction Endonucleases has elicited many hypotheses, differing mainly in the number of Mg(2+) involved in catalysis. To address this problem, we measured the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me(2+) per active site. DNA cleavage experiments were carried out at various Mg(2+) and Mn(2+) concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg(2+) and Mn(2+) concentration dependence. In general, the Mg(2+) concentration optimum (between approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 0.1 and 1 mM). At still higher Mg(2+) or Mn(2+) concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca(2+). Based on these results, we propose that one Mg(2+) or Mn(2+) is critical for Restriction enzyme activation, and binding of a second Me(2+) plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg(2+) or Mn(2+) mainly leads to an increase in K(m), such that the inhibitory effect of excess Mg(2+) or Mn(2+) can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me(2+) binding to these enzymes.
-
type ii Restriction Endonucleases structure and mechanism
Cellular and Molecular Life Sciences, 2005Co-Authors: Alfred Pingoud, Monika Fuxreiter, Vera Pingoud, Wolfgang WendeAbstract:Type II Restriction Endonucleases are components of Restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few Restriction Endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II Restriction Endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II Restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.
