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R. Stephen Lloyd - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of DNA Glycosylases via small molecule purine analogs.
PLOS ONE, 2013Co-Authors: Aaron C. Jacobs, Amanda K. Mccullough, Marcus J. Calkins, Ajit Jadhav, Dorjbal Dorjsuren, David G. Maloney, Anton Simeonov, Pawel Jaruga, Miral Dizdaroglu, R. Stephen LloydAbstract:Following the formation of oxidatively-induced DNA damage, several DNA Glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA Glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA Glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA Glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle Glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined Glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA Glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific Glycosylase.
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TAT-mediated delivery of a DNA repair enzyme to skin cells rapidly initiates repair of UV-induced DNA damage
Journal of Investigative Dermatology, 2011Co-Authors: Jodi L. Johnson, Brian C. Lowell, Olga P. Ryabinina, R. Stephen Lloyd, Amanda K. McculloughAbstract:UV light causes DNA damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed annually in the United States. Although human cells possess a mechanism (nucleotide excision repair) to repair UV-induced DNA damage, mutagenesis still occurs when DNA is replicated before repair of these photoproducts. Although human cells have all the enzymes necessary to complete an alternate repair pathway, base excision repair (BER), they lack a DNA Glycosylase that can initiate BER of dipyrimidine photoproducts. Certain prokaryotes and viruses produce pyrimidine dimer-specific DNA Glycosylases (pdgs) that initiate BER of cyclobutane pyrimidine dimers (CPDs), the predominant UV-induced lesions. Such a pdg was identified in the Chlorella virus PBCV-1 and termed Cv-pdg. The Cv-pdg protein was engineered to contain a nuclear localization sequence (NLS) and a membrane permeabilization peptide (transcriptional transactivator, TAT). Here, we demonstrate that the Cv-pdg-NLS-TAT protein was delivered to repair-proficient keratinocytes and fibroblasts, and to a human skin model, where it rapidly initiated removal of CPDs. These data suggest a potential strategy for prevention of human skin cancer.
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Human Polymorphic Variants of the NEIL1 DNA Glycosylase
Journal of Biological Chemistry, 2007Co-Authors: Pawel Jaruga, Amanda K. Mccullough, Miral Dizdaroglu, Thomas G. Wood, R. Stephen LloydAbstract:Abstract In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA Glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864-1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type β,δ-elimination reaction but primarily yielded the β-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and γ-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of Glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be Glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome.
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Mechanistic comparisons among base excision repair Glycosylases.
Free Radical Biology and Medicine, 2002Co-Authors: M L Dodson, R. Stephen LloydAbstract:Abstract The mechanisms by which various DNA Glycosylases initiate the base excision repair pathways are discussed. Fundamental distinctions are made between “simple Glycosylases,” that do not form DNA single-strand breaks, and “Glycosylases/abasic site lyases,” that do form single-strand breaks. Several groupings of BER substrate sites are defined and some interactions between these groupings and Glycosylase mechanisms discussed. Two characteristics are proposed to be common among all BER Glycosylases: a nucleotide flipping step that serves to expose the scissile glycosyl bond to catalysis, and a Glycosylase transition state characterized by substantial tetrahedral character at the base glycosyl atom.
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Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer Glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific Glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific Glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for Glycosylase/AP lyase enzymes in which the Glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.
A K McCullough - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer Glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific Glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific Glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for Glycosylase/AP lyase enzymes in which the Glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.
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Characterization of a Novel Cis-Syn and Trans-Syn-Ii Pyrimidinedimer Glycosylase Ap Lyase From a Eukaryotic Algal Virus,Paramecium Bursaria Chlorella Virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidinedimer-specific Glycosylase, Recently, the first sequencehomolog of T4 endonuclease V was identified from chlorellavirus Paramecium bursaria chlorella virus-1 (PBCV-1). Here wepresent the biochemical characterization of the chlorella viruspyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG isspecific not only for the cis-syn cyclobutane pyrimidine dimer,but also for the trans-syn-II isomer, This is the first trans-syn-II-specific Glycosylase identified to date. Kineticanalysis demonstrates that DNAs containing both types ofpyrimidine dimers are cleaved by the enzyme with similarcatalytic efficiencies. Cleavage analysis and covalent trappingexperiments demonstrate that the enzyme mechanism is consistentwith the model proposed for Glycosylase/AP lyase enzymes inwhich the Glycosylase action is mediated via an iminointermediate between the C1' of the sugar and an amino group inthe enzyme, followed by a beta-elimination reaction resultingin cleavage of the phosphodiester bond. cv-PDG exhibitsprocessive cleavage kinetics which are diminished at saltconcentrations greater than those determined for T4endonuclease V, indicating a possibly stronger electrostaticattraction between enzyme and DNA. The identification of thisnew enzyme with broader pyrimidine dimer specificity raises theintriguing possibility that there may be other T4 endonucleaseV-like enzymes with specificity toward other DNA photoproducts.
M L Dodson - One of the best experts on this subject based on the ideXlab platform.
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Modulation of the turnover of formamidopyrimidine DNA Glycosylase
Biochemistry, 2006Co-Authors: Michael B. Harbut, M L Dodson, Michael G. Meador, Robert G. LloydAbstract:Prokaryotic and eukaryotic cells utilize a number of different mechanisms to repair constantly accumulating DNA damage due to environmental and endogenous chemical agents. In the absence of repair, many DNA lesions may block replication and transcription, while others may decrease replication fidelity. These disruptions may result in mutations and ultimately carcinogenesis in eukaryotes. The base excision repair (BER)1 pathway is a key component in the cellular response to DNA lesions (1). Formamidopyrimidine DNA Glycosylase (Fpg) functions in the E. coli BER pathway as an N-Glycosylase and abasic site (AP) lyase, with the predominant DNA incision product resulting from a δ-elimination reaction (2). The Fpg Glycosylase activity results in the removal of formamidopyrimidine (Fapy) or 8-oxo-7,8-dihydroguanine (8-oxoG) lesions (3, 4). The catalytic mechanism of Fpg is similar to that of other BER Glycosylases that manifest an AP lyase capability (5). The active site nucleophile is the N-terminal proline secondary amine (6). This nucleophile collapses onto C1′ of the scissile base deoxyribose moiety due to the developing electrophilic character at C1′, concomitant with the Glycosylase step (5, Dodson, unpublished). The crystal and cocrystal structures of the E. coli Fpg and Fpg homologues from other bacterial and human sources have been published (7–11). Recent NMR analyses of free Fpg and Fpg bound to duplex DNA containing a site-specific AP site analogue, revealed a highly dynamic structure, even in complex with DNA (12). These data are consistent with molecular dynamics calculations of the enzyme in complex with 8-oxoG, even though the conclusions of these investigations differed concerning the anti/syn conformation of a bound 8-oxoG substrate (13–15). Precatalytic and catalytic mechanisms of Fpg have also been extensively investigated (13, 16–20). These investigations reveal a complex series of enzyme and DNA conformational changes that ultimately result in the formation of the Michaelis complex. Predictions concerning amino acid residues that could modulate precatalytic and catalytic steps based on structures, dynamics and spectroscopy have been successfully carried out, revealing molecular details of the overall catalytic mechanism (13). McCullough et al. (5) have discussed that the product spectra of the BER Glycosylase-lyase enzymes arise as a consequence of kinetic competitions between sequential elimination events (glycosyl bond cleavage, β-elimination, δ-elimination) and hydrolysis of the corresponding covalent enzyme-product intermediates. Bhagwat and Gerlt (21) demonstrated that the Fpg δ-elimination step is dependent on a prior β-elimination step, a result that supports the kinetic competition argument. One corollary of that argument is that the cleavage of the covalent product from the enzyme is rate limiting in the catalytic scheme. The predominance of the δ-elimination product is consistent with the slow hydrolysis of Schiff base intermediates involving secondary amines. It has been suggested that the conserved Glu3 residue electrostatically stabilizes the electrophilic C1′ and subsequently, the protonated Schiff base intermediate (22, 23). Mutation of Lys57, whose e-amine contacts the phosphates one and two nucleotides 3′ to the 8-oxoG site, abolishes activity on 8-oxoG, but has little effect on AP lyase activity (8, 24). Gilboa et al. (8) have suggested that Lys57 protonates the leaving 3′ phosphate, thereby accelerating the β-elimination step. Results from studies of other combined Glycosylase-AP lyases, such as T4 pyrimidine dimer Glycosylase (T4-Pdg) and endonuclease VIII may also be used to suggest key amino acid residues involved in the catalytic mechanism of Fpg, due to a commonality in catalytic mechanisms. Meador et al. (25) showed that mutation of the His16 residue of T4-Pdg resulted in an enzyme that was severely compromised in turnover following the catalytic events. The His16 residue had been originally hypothesized to be involved in the formation of the Schiff base or subsequent intermediates. Further investigations did not support this hypothesis. The mutant enzymes were able to carry out the Glycosylase step, form the Schiff base intermediate, and carry out the lyase step. However, the rate of product formation rapidly plateaued over time and was directly proportional to the amount of enzyme added. It was concluded that the His16 residue was involved in enzyme-product turnover. The mechanistic similarities between T4-Pdg and Fpg suggested that some residue in the Fpg structure might play a similar role to that of T4-Pdg His16. This study tests that hypothesis by identifying a similar candidate histidine residue, mutating that residue and investigating the alteration in catalytic properties of the mutant enzymes. The commonality of mutant enzyme phenotypes exists despite a complete lack of structural or sequence homology and entirely different active site architectures. The implication is that these residues catalyze the rate limiting and essential step for these BER DNA lyases.
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Mechanistic comparisons among base excision repair Glycosylases.
Free Radical Biology and Medicine, 2002Co-Authors: M L Dodson, R. Stephen LloydAbstract:Abstract The mechanisms by which various DNA Glycosylases initiate the base excision repair pathways are discussed. Fundamental distinctions are made between “simple Glycosylases,” that do not form DNA single-strand breaks, and “Glycosylases/abasic site lyases,” that do form single-strand breaks. Several groupings of BER substrate sites are defined and some interactions between these groupings and Glycosylase mechanisms discussed. Two characteristics are proposed to be common among all BER Glycosylases: a nucleotide flipping step that serves to expose the scissile glycosyl bond to catalysis, and a Glycosylase transition state characterized by substantial tetrahedral character at the base glycosyl atom.
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initiation of base excision repair Glycosylase mechanisms and structures
Annual Review of Biochemistry, 1999Co-Authors: Amanda K. Mccullough, M L Dodson, Robert LloydAbstract:▪ Abstract The base excision repair pathway is an organism's primary defense against mutations induced by oxidative, alkylating, and other DNA-damaging agents. This pathway is initiated by DNA Glycosylases that excise the damaged base by cleavage of the glycosidic bond between the base and the DNA sugar-phosphate backbone. A subset of Glycosylases has an associated apurinic/apyrimidinic (AP) lyase activity that further processes the AP site to generate cleavage of the DNA phosphate backbone. Chemical mechanisms that are supported by biochemical and structural data have been proposed for several Glycosylases and Glycosylase/AP lyases. This review focuses on the chemical mechanisms of catalysis in the context of recent structural information, with emphasis on the catalytic residues and the active site conformations of several cocrystal structures of Glycosylases with their substrate DNAs. Common structural motifs for DNA binding and damage specificity as well as conservation of acidic residues and amino gro...
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Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer Glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific Glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific Glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for Glycosylase/AP lyase enzymes in which the Glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.
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Characterization of a Novel Cis-Syn and Trans-Syn-Ii Pyrimidinedimer Glycosylase Ap Lyase From a Eukaryotic Algal Virus,Paramecium Bursaria Chlorella Virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidinedimer-specific Glycosylase, Recently, the first sequencehomolog of T4 endonuclease V was identified from chlorellavirus Paramecium bursaria chlorella virus-1 (PBCV-1). Here wepresent the biochemical characterization of the chlorella viruspyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG isspecific not only for the cis-syn cyclobutane pyrimidine dimer,but also for the trans-syn-II isomer, This is the first trans-syn-II-specific Glycosylase identified to date. Kineticanalysis demonstrates that DNAs containing both types ofpyrimidine dimers are cleaved by the enzyme with similarcatalytic efficiencies. Cleavage analysis and covalent trappingexperiments demonstrate that the enzyme mechanism is consistentwith the model proposed for Glycosylase/AP lyase enzymes inwhich the Glycosylase action is mediated via an iminointermediate between the C1' of the sugar and an amino group inthe enzyme, followed by a beta-elimination reaction resultingin cleavage of the phosphodiester bond. cv-PDG exhibitsprocessive cleavage kinetics which are diminished at saltconcentrations greater than those determined for T4endonuclease V, indicating a possibly stronger electrostaticattraction between enzyme and DNA. The identification of thisnew enzyme with broader pyrimidine dimer specificity raises theintriguing possibility that there may be other T4 endonucleaseV-like enzymes with specificity toward other DNA photoproducts.
Simon Nyaga - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer Glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific Glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific Glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for Glycosylase/AP lyase enzymes in which the Glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.
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Characterization of a Novel Cis-Syn and Trans-Syn-Ii Pyrimidinedimer Glycosylase Ap Lyase From a Eukaryotic Algal Virus,Paramecium Bursaria Chlorella Virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidinedimer-specific Glycosylase, Recently, the first sequencehomolog of T4 endonuclease V was identified from chlorellavirus Paramecium bursaria chlorella virus-1 (PBCV-1). Here wepresent the biochemical characterization of the chlorella viruspyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG isspecific not only for the cis-syn cyclobutane pyrimidine dimer,but also for the trans-syn-II isomer, This is the first trans-syn-II-specific Glycosylase identified to date. Kineticanalysis demonstrates that DNAs containing both types ofpyrimidine dimers are cleaved by the enzyme with similarcatalytic efficiencies. Cleavage analysis and covalent trappingexperiments demonstrate that the enzyme mechanism is consistentwith the model proposed for Glycosylase/AP lyase enzymes inwhich the Glycosylase action is mediated via an iminointermediate between the C1' of the sugar and an amino group inthe enzyme, followed by a beta-elimination reaction resultingin cleavage of the phosphodiester bond. cv-PDG exhibitsprocessive cleavage kinetics which are diminished at saltconcentrations greater than those determined for T4endonuclease V, indicating a possibly stronger electrostaticattraction between enzyme and DNA. The identification of thisnew enzyme with broader pyrimidine dimer specificity raises theintriguing possibility that there may be other T4 endonucleaseV-like enzymes with specificity toward other DNA photoproducts.
M T Romberg - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer Glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific Glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific Glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for Glycosylase/AP lyase enzymes in which the Glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.
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Characterization of a Novel Cis-Syn and Trans-Syn-Ii Pyrimidinedimer Glycosylase Ap Lyase From a Eukaryotic Algal Virus,Paramecium Bursaria Chlorella Virus-1
Journal of Biological Chemistry, 1998Co-Authors: A K McCullough, Simon Nyaga, M T Romberg, Y F Wei, T G Wood, M L Dodson, James L. Van Etten, J. S. Taylor, R. Stephen LloydAbstract:Endonuclease V from bacteriophage T4, is a cis-syn pyrimidinedimer-specific Glycosylase, Recently, the first sequencehomolog of T4 endonuclease V was identified from chlorellavirus Paramecium bursaria chlorella virus-1 (PBCV-1). Here wepresent the biochemical characterization of the chlorella viruspyrimidine dimer Glycosylase, cv-PDG. Interestingly, cv-PDG isspecific not only for the cis-syn cyclobutane pyrimidine dimer,but also for the trans-syn-II isomer, This is the first trans-syn-II-specific Glycosylase identified to date. Kineticanalysis demonstrates that DNAs containing both types ofpyrimidine dimers are cleaved by the enzyme with similarcatalytic efficiencies. Cleavage analysis and covalent trappingexperiments demonstrate that the enzyme mechanism is consistentwith the model proposed for Glycosylase/AP lyase enzymes inwhich the Glycosylase action is mediated via an iminointermediate between the C1' of the sugar and an amino group inthe enzyme, followed by a beta-elimination reaction resultingin cleavage of the phosphodiester bond. cv-PDG exhibitsprocessive cleavage kinetics which are diminished at saltconcentrations greater than those determined for T4endonuclease V, indicating a possibly stronger electrostaticattraction between enzyme and DNA. The identification of thisnew enzyme with broader pyrimidine dimer specificity raises theintriguing possibility that there may be other T4 endonucleaseV-like enzymes with specificity toward other DNA photoproducts.
