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Janusz M Bujnicki - One of the best experts on this subject based on the ideXlab platform.
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phylogenomics and sequence structure function relationships in the gmrsd family of type iv Restriction Enzymes
BMC Bioinformatics, 2015Co-Authors: Magdalena A Machnicka, Janusz M Bujnicki, Katarzyna Kaminska, Stanislaw DuninhorkawiczAbstract:GmrSD is a modification-dependent Restriction endonuclease that specifically targets and cleaves glucosylated hydroxymethylcytosine (glc-HMC) modified DNA. It is encoded either as two separate single-domain GmrS and GmrD proteins or as a single protein carrying both domains. Previous studies suggested that GmrS acts as endonuclease and NTPase whereas GmrD binds DNA. In this work we applied homology detection, sequence conservation analysis, fold recognition and homology modeling methods to study sequence-structure-function relationships in the GmrSD Restriction endonucleases family. We also analyzed the phylogeny and genomic context of the family members. Results of our comparative genomics study show that GmrS exhibits similarity to proteins from the ParB/Srx fold which can have both NTPase and nuclease activity. In contrast to the previous studies though, we attribute the nuclease activity also to GmrD as we found it to contain the HNH endonuclease motif. We revealed residues potentially important for structure and function in both domains. Moreover, we found that GmrSD systems exist predominantly as a fused, double-domain form rather than as a heterodimer and that their homologs are often encoded in regions enriched in defense and gene mobility-related elements. Finally, phylogenetic reconstructions of GmrS and GmrD domains revealed that they coevolved and only few GmrSD systems appear to be assembled from distantly related GmrS and GmrD components. Our study provides insight into sequence-structure-function relationships in the yet poorly characterized family of Type IV Restriction Enzymes. Comparative genomics allowed to propose possible role of GmrD domain in the function of the GmrSD enzyme and possible active sites of both GmrS and GmrD domains. Presented results can guide further experimental characterization of these Enzymes.
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Phylogenomics and sequence-structure-function relationships in the GmrSD family of Type IV Restriction Enzymes
BMC Bioinformatics, 2015Co-Authors: Magdalena A Machnicka, Katarzyna H. Kaminska, Stanislaw Dunin-horkawicz, Janusz M BujnickiAbstract:Background GmrSD is a modification-dependent Restriction endonuclease that specifically targets and cleaves glucosylated hydroxymethylcytosine (glc-HMC) modified DNA. It is encoded either as two separate single-domain GmrS and GmrD proteins or as a single protein carrying both domains. Previous studies suggested that GmrS acts as endonuclease and NTPase whereas GmrD binds DNA. Methods In this work we applied homology detection, sequence conservation analysis, fold recognition and homology modeling methods to study sequence-structure-function relationships in the GmrSD Restriction endonucleases family. We also analyzed the phylogeny and genomic context of the family members. Results Results of our comparative genomics study show that GmrS exhibits similarity to proteins from the ParB/Srx fold which can have both NTPase and nuclease activity. In contrast to the previous studies though, we attribute the nuclease activity also to GmrD as we found it to contain the HNH endonuclease motif. We revealed residues potentially important for structure and function in both domains. Moreover, we found that GmrSD systems exist predominantly as a fused, double-domain form rather than as a heterodimer and that their homologs are often encoded in regions enriched in defense and gene mobility-related elements. Finally, phylogenetic reconstructions of GmrS and GmrD domains revealed that they coevolved and only few GmrSD systems appear to be assembled from distantly related GmrS and GmrD components. Conclusions Our study provides insight into sequence-structure-function relationships in the yet poorly characterized family of Type IV Restriction Enzymes. Comparative genomics allowed to propose possible role of GmrD domain in the function of the GmrSD enzyme and possible active sites of both GmrS and GmrD domains. Presented results can guide further experimental characterization of these Enzymes.
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structural and evolutionary classification of type ii Restriction Enzymes based on theoretical and experimental analyses
Nucleic Acids Research, 2008Co-Authors: Jerzy Orlowski, Janusz M BujnickiAbstract:For a very long time, Type II Restriction Enzymes (REases) have been a paradigm of ORFans: proteins with no detectable similarity to each other and to any other protein in the database, despite common cellular and biochemical function. Crystallographic analyses published until January 2008 provided high-resolution structures for only 28 of 1637 Type II REase sequences available in the Restriction Enzyme database (REBASE). Among these structures, all but two possess catalytic domains with the common PD-(D/E)XK nuclease fold. Two structures are unrelated to the others: R.BfiI exhibits the phospholipase D (PLD) fold, while R.PabI has a new fold termed ‘half-pipe’. Thus far, bioinformatic studies supported by site-directed mutagenesis have extended the number of tentatively assigned REase folds to five (now including also GIY-YIG and HNH folds identified earlier in homing endonucleases) and provided structural predictions for dozens of REase sequences without experimentally solved structures. Here, we present a comprehensive study of all Type II REase sequences available in REBASE together with their homologs detectable in the nonredundant and environmental samples databases at the NCBI. We present the summary and critical evaluation of structural assignments and predictions reported earlier, new classification of all REase sequences into families, domain architecture analysis and new predictions of three-dimensional folds. Among 289 experimentally characterized (not putative) Type II REases, whose apparently full-length sequences are available in REBASE, we assign 199 (69%) to contain the PD-(D/E)XK domain. The HNH domain is the second most common, with 24 (8%) members. When putative REases are taken into account, the fraction of PD-(D/E)XK and HNH folds changes to 48% and 30%, respectively. Fifty-six characterized (and 521 predicted) REases remain unassigned to any of the five REase folds identified so far, and may exhibit new architectures. These Enzymes are proposed as the most interesting targets for structure determination by high-resolution experimental methods. Our analysis provides the first comprehensive map of sequence-structure relationships among Type II REases and will help to focus the efforts of structural and functional genomics of this large and biotechnologically important class of Enzymes.
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novel protein fold discovered in the pabi family of Restriction Enzymes
Nucleic Acids Research, 2007Co-Authors: Kenichi Miyazono, Janusz M Bujnicki, Miki Watanabe, Jan Kosinski, Ken Ishikawa, Masayuki Kamo, Tatsuya Sawasaki, Koji Nagata, Yaeta Endo, Masaru TanokuraAbstract:Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II Restriction–modification gene complexes as mobile elements, our earlier work identified a Restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a ‘half pipe’. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most Restriction Enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg2+. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.
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specificity changes in the evolution of type ii Restriction endonucleases a biochemical and bioinformatic analysis of Restriction Enzymes that recognize unrelated sequences
Journal of Biological Chemistry, 2005Co-Authors: Vera Pingoud, Janusz M Bujnicki, Anna Sudina, Hildegard Geyer, Rudi Lurz, Gerhild Luder, Richard D Morgan, E A Kubareva, Alfred PingoudAbstract:Abstract How Restriction Enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II Restriction endonucleases, namely SsoII (↓CCNGG), PspGI (↓CCWGG), Eco-RII (↓CCWGG), NgoMIV (G↓CCGGC), and Cfr10I (R↓CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of ∼70 amino acid residues with MboI, a Type II Restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913–929). Nevertheless, MboI has a dissimilar DNA specificity (↓GATC) compared with these Enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of ∼70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II Restriction Enzymes of widely different specificities.
Virginijus Siksnys - One of the best experts on this subject based on the ideXlab platform.
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Time-resolved fluorescence studies of nucleotide flipping by Restriction Enzymes
Nucleic Acids Research, 2009Co-Authors: Robert K Neely, Marta Kubala, Virginijus Siksnys, Gintautas Tamulaitis, Kai Chen, Anita C JonesAbstract: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.
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tetrameric Restriction Enzymes expansion to the giy yig nuclease family
Nucleic Acids Research, 2007Co-Authors: Giedrius Gasiunas, Gintautas Tamulaitis, Giedrius Sasnauskas, Claus Urbanke, Dalia Razaniene, Virginijus SiksnysAbstract: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.
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biochemical and mutational analysis of ecorii functional domains reveals evolutionary links between Restriction Enzymes
FEBS Letters, 2006Co-Authors: Gintautas Tamulaitis, Merlind Mucke, Virginijus SiksnysAbstract: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.
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structure and function of the tetrameric Restriction Enzymes
2004Co-Authors: Virginijus Siksnys, S Grazulis, Robert HuberAbstract:Type II Restriction endonucleases recognize specific DNA sequences, typically 4–8 bp in length, and cleave phosphodiester bonds in the presence of Mg2+, within or close to their recognition sites (Pingoud and Jeltsch 2001). Around 3500 species, from variety of bacteria with nearly 240 differing specificities, have now been characterized (Roberts et al. 2003). Most of the sequences recognized by Type II Restriction endonucleases are palindromic, i. e., possess a twofold rotational axis of symmetry. On the basis of this observation, Kelly and Smith proposed the first model for the interaction of these Restriction Enzymes with DNA (Kelly and Smith 1970).According to their model (Fig. 1), recognition of the palindromic DNA sequence is achieved by two identical protein subunits related by a twofold axis of symmetry. Each subunit faces the same nucleotide sequence on the opposite DNA strand and contains one active site. Symmetrical nicking of opposite DNA strands by both monomers within a homodimer generates the double-strand break. Hence, the symmetry of the recognition sequence implies the oligomeric state of the Restriction enzyme.
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how the bfii Restriction enzyme uses one active site to cut two dna strands
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Giedrius Sasnauskas, Stephen E Halford, Virginijus SiksnysAbstract:Unlike other Restriction Enzymes, BfiI functions without metal ions. It recognizes an asymmetric DNA sequence, 5′-ACTGGG-3′, and cuts top and bottom strands at fixed positions downstream of this sequence. Many Restriction Enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like FokI, dimerize transiently during catalysis. BfiI is also a dimer but it has only one active site, at the dimer interface. We show here that BfiI remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and/or the DNA in the BfiI–DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting BfiI into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5′ phosphate in the bottom strand. BfiI thus uses a mechanism for making double-strand breaks that is novel among Restriction Enzymes.
David T F Dryden - One of the best experts on this subject based on the ideXlab platform.
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structures of the type i dna Restriction Enzymes
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: David T F DrydenAbstract:The article by Liu et al. (1) on the structure of type I DNA Restriction and modification Enzymes purports to significantly advance our understanding of these Enzymes and proposes a model for their operation. While the partial structure of one of these Enzymes is interesting and defines the interface between some of the subunits, the article contains many misinterpretations of the literature. In 1968, these Enzymes were the first Restriction Enzymes to be purified (2). It was soon apparent that they contained three subunits; R, M, and S for Restriction, modification, and sequence specificity, respectively, in the complex R2M2S1. A M2S1 complex could act … [↵][1]1Email: david.t.dryden{at}durham.ac.uk. [1]: #xref-corresp-1-1
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highlights of the dna cutters a short history of the Restriction Enzymes
Nucleic Acids Research, 2014Co-Authors: Wil A M Loenen, David T F Dryden, Elisabeth A Raleigh, Geoffrey G Wilson, Noreen E. MurrayAbstract:In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA Restriction-modification systems. The Type II Restriction Enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of Restriction Enzymes and their continuing impact on molecular biology and medicine.
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type i Restriction Enzymes and their relatives
Nucleic Acids Research, 2014Co-Authors: Wil A M Loenen, David T F Dryden, Elisabeth A Raleigh, Geoffrey G WilsonAbstract:Type I Restriction Enzymes (REases) are large pentameric proteins with separate Restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I Enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the Enzymes and of their S subunits. Finally, we describe examples of Type II Restriction-modification systems that have features in common with Type I Enzymes, with emphasis on the varied Type IIG Enzymes.
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DNA translocation by type III Restriction Enzymes: A comparison of current models of their operation derived from ensemble and single-molecule measurements
Nucleic Acids Research, 2011Co-Authors: David T F Dryden, J. Michael Edwardson, Robert M HendersonAbstract:Much insight into the interactions of DNA and Enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques-atomic force microscopy (AFM) and magnetic tweezers (MT)-have produced apparently contradictory results when applied to the action of the ATP-dependent type III Restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme-enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this 'Survey and Summary', we describe several different models put forward for the action of type III Restriction Enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these Enzymes.
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dna looping and translocation provide an optimal cleavage mechanism for the type iii Restriction Enzymes
The EMBO Journal, 2007Co-Authors: Neal Crampton, David T F Dryden, Desirazu N Rao, Stefanie Roes, Michael J Edwardson, Robert M HendersonAbstract:EcoP15I is a type III Restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I Enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I–DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for Restriction by type III Restriction Enzymes and highlight the similarities between this and other classes of Restriction Enzymes.
Desirazu N Rao - One of the best experts on this subject based on the ideXlab platform.
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mechanistic insights into type iii Restriction Enzymes
Frontiers in Bioscience, 2012Co-Authors: Nidhanapati K Raghavendra, Shivakumara Bheemanaik, Desirazu N RaoAbstract:Type III Restriction-modification (R-M) Enzymes need to interact with two separate unmethylated DNA sequences in indirectly repeated, head-to-head orientations for efficient cleavage to occur at a defined location next to only one of the two sites. However, cleavage of sites that are not in head-to-head orientation have been observed to occur under certain reaction conditions in vitro. ATP hydrolysis is required for the long-distance communication between the sites prior to cleavage. Type III R-M Enzymes comprise two subunits, Res and Mod that form a homodimeric Mod2 and a heterotetrameric Res2Mod2 complex. The Mod subunit in M2 or R2M2 complex recognizes and methylates DNA while the Res subunit in R2M2 complex is responsible for ATP hydrolysis, DNA translocation and cleavage. A vast majority of biochemical studies on Type III R-M Enzymes have been undertaken using two closely related Enzymes, EcoP1I and EcoP15I. Divergent opinions about how the long-distance interaction between the recognition sites exist and at least three mechanistic models based on 1D- diffusion and/or 3D- DNA looping have been proposed.
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dna looping and translocation provide an optimal cleavage mechanism for the type iii Restriction Enzymes
The EMBO Journal, 2007Co-Authors: Neal Crampton, David T F Dryden, Desirazu N Rao, Stefanie Roes, Michael J Edwardson, Robert M HendersonAbstract:EcoP15I is a type III Restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I Enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I–DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for Restriction by type III Restriction Enzymes and highlight the similarities between this and other classes of Restriction Enzymes.
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s adenosyl l methionine dependent Restriction Enzymes
Critical Reviews in Biochemistry and Molecular Biology, 2004Co-Authors: Srivani Sistla, Desirazu N RaoAbstract:Restriction-modification (R-M) Enzymes are classified into type I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. While the role of S-Adenosy-L-methionine (AdoMet) as the methyl group donor in the methylation reaction is undisputed, its requirement in DNA cleavage reaction has been subject to intense study. AdoMet is a prerequisite for the DNA cleavage by most type I Enzymes known so far, with the exception of R.EcoR124I. A number of new type II Restriction Enzymes belonging to the type IIB and IIG family were found to show AdoMet dependence for their cleavage reaction. The type III Enzymes have been found to require AdoMet for their Restriction function. Ado Met functions as an allosteric effector of the DNA cleavage reaction and has been shown to bring about conformational changes in the protein upon binding.
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nucleoside triphosphate dependent Restriction Enzymes
Nucleic Acids Research, 2001Co-Authors: David T F Dryden, Noreen E. Murray, Desirazu N RaoAbstract:The known nucleoside triphosphate-dependent Restriction Enzymes are hetero-oligomeric proteins that behave as molecular machines in response to their target sequences. They translocate DNA in a process dependent on the hydrolysis of a nucleoside triphosphate. For the ATP-dependent type I and type III Restriction and modification systems, the collision of translocating complexes triggers hydrolysis of phosphodiester bonds in unmodified DNA to generate double-strand breaks. Type I endonucleases break the DNA at unspecified sequences remote from the target sequence, type III endonucleases at a fixed position close to the target sequence. Type I and type III Restriction and modification (R-M) systems are notable for effective post-translational control of their endonuclease activity. For some type I Enzymes, this control is mediated by proteolytic degradation of that subunit of the complex which is essential for DNA translocation and breakage. This control, lacking in the well-studied type II R-M systems, provides extraordinarily effective protection of resident DNA should it acquire unmodified target sequences. The only well-documented GTP-dependent Restriction enzyme, McrBC, requires methylated target sequences for the initiation of phosphodiester bond cleavage.
Robert M Henderson - One of the best experts on this subject based on the ideXlab platform.
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DNA translocation by type III Restriction Enzymes: A comparison of current models of their operation derived from ensemble and single-molecule measurements
Nucleic Acids Research, 2011Co-Authors: David T F Dryden, J. Michael Edwardson, Robert M HendersonAbstract:Much insight into the interactions of DNA and Enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques-atomic force microscopy (AFM) and magnetic tweezers (MT)-have produced apparently contradictory results when applied to the action of the ATP-dependent type III Restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme-enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this 'Survey and Summary', we describe several different models put forward for the action of type III Restriction Enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these Enzymes.
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dna looping and translocation provide an optimal cleavage mechanism for the type iii Restriction Enzymes
The EMBO Journal, 2007Co-Authors: Neal Crampton, David T F Dryden, Desirazu N Rao, Stefanie Roes, Michael J Edwardson, Robert M HendersonAbstract:EcoP15I is a type III Restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I Enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I–DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for Restriction by type III Restriction Enzymes and highlight the similarities between this and other classes of Restriction Enzymes.
