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Stephen R Lloyd - One of the best experts on this subject based on the ideXlab platform.
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uncoupling of nucleotide flipping and dna bending by the t4 Pyrimidine Dimer dna glycosylase
Biochemistry, 2006Co-Authors: Randall K Walker, Amanda K Mccullough, Stephen R LloydAbstract:Bacteriophage T4 Pyrimidine Dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane Pyrimidine Dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5'-thymine of a thymine-thymine (TT) Dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60 degrees in the enzyme-substrate (ES) complex. To examine the kinetic details of the precatalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately assess nucleotide flipping and DNA bending. The fluorescent adenine base analogue, 2-aminopurine (2-AP), placed opposite an abasic site analogue, tetrahydrofuran, exhibited a 2.8-fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping, kon and koff pre-steady-state kinetic measurements were determined. DNA bending was assessed by fluorescence resonance energy transfer using fluorescent donor-acceptor pairs located at the 5'-ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5-fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.
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structure of t4 Pyrimidine Dimer glycosylase in a reduced imine covalent complex with abasic site containing dna
Journal of Molecular Biology, 2006Co-Authors: Gali Golan, Amanda K Mccullough, Stephen R Lloyd, M L Dodson, Dmitry O Zharkov, Arthur P Grollman, Gil ShohamAbstract:Abstract The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane Pyrimidine Dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 Pyrimidine Dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the α-amino group of the N-terminal threonine residue and the C1′ of the deoxyribose sugar of the 5′ Pyrimidine at the Dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA–protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing Pyrimidine Dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (∼ 66°) in the DNA at the Dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane Dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.
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investigations of Pyrimidine Dimer glycosylases a paradigm for dna base excision repair enzymology
Mutation Research, 2005Co-Authors: Stephen R LloydAbstract:The most prevalent forms of cancer in humans are the non-melanoma skin cancers, with over a million new cases diagnosed in the United States annually. The portions of the body where these cancers arise are almost exclusively on the most heavily sun-exposed tissues. It is now well established that exposure to ultraviolet light (UV) causes not only damage to DNA that subsequently generates mutations and a transformed phenotype, but also UV-induced immunosuppression. Human cells have only one mechanism to remove the UV-induced diPyrimidine DNA photoproducts: nucleotide excision repair (NER). However, simpler organisms such as bacteria, bacteriophages and some eukaryotic viruses contain up to three distinct mechanisms to initiate the repair of UV-induced diPyrimidine adducts: NER, base excision repair (BER) and photoreversal. This review will focus on the biology and the mechanisms of DNA glycosylase/AP lyases that initiate BER of cis-syn cyclobutane Pyrimidine Dimers. One of these enzymes, the T4 Pyrimidine Dimer glycosylase (T4-pdg), formerly known as T4 endonuclease V has served as a model in the study of this entire class of enzymes. It was the first DNA repair enzyme: (1) for which a biologically significant processive nicking activity was demonstrated; (2) to have its active site determined, (3) to have its crystal structure solved, (4) to be shown to carry out nucleotide flipping, and (5) to be used in human clinical trials for disease prevention.
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role of his 16 in turnover of t4 Pyrimidine Dimer glycosylase
Journal of Biological Chemistry, 2004Co-Authors: Michael G Meador, Stephen R Lloyd, Lavanya Rajagopalan, M L DodsonAbstract:Abstract Previously, the histidine residue at position 16 in the mature T4 Pyrimidine Dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle.
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chlorella virus Pyrimidine Dimer glycosylase excises ultraviolet radiation and hydroxyl radical induced products 4 6 diamino 5 formamidoPyrimidine and 2 6 diamino 4 hydroxy 5 formamidoPyrimidine from dna
Photochemistry and Photobiology, 2002Co-Authors: Pawel Jaruga, Ritche Jabil, Amanda K Mccullough, Henry Rodriguez, Miral Dizdaroglu, Stephen R LloydAbstract:A DNA glycosylase specific for UV radiation‐induced Pyrimidine Dimers has been identified from the Chlorella virus Paramecium Bursaria Chlorella virus-1. This enzyme (Chlorella virus Pyrimidine Dimer glycosylase [cvpdg]) exhibits a 41% amino acid identity with endonuclease V from bacteriophage T4 (T4 Pyrimidine Dimer glycosylase [T4-pdg]), which is also specific for Pyrimidine Dimers. However, cv-pdg possesses a higher catalytic efficiency and broader substrate specificity than T4-pdg. The latter excises 4,6-diamino-5-formamidoPyrimidine (FapyAde), a UV radiation‐ and hydroxyl radical‐induced monomeric product of adenine in DNA. Using gas chromatography‐isotope-dilution mass spectrometry and g-irradiated DNA, we show in this work that cv-pdg also displays a catalytic activity for excision of FapyAde and, in addition, it excises 2,6-diamino-4-hydroxy-5-formamidoPyrimidine (FapyGua). Kinetic data show that FapyAde is a better substrate for cv-pdg than FapyGua. On the other hand, cv-pdg possesses a greater efficiency
Shigenori Iwai - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the nucleosome containing ultraviolet light induced cyclobutane Pyrimidine Dimer
Biochemical and Biophysical Research Communications, 2016Co-Authors: Naoki Horikoshi, Shigenori Iwai, Hiroaki Tachiwana, Wataru Kagawa, Akihisa Osakabe, Syota Matsumoto, Kaoru Sugasawa, Hitoshi KurumizakaAbstract:The cyclobutane Pyrimidine Dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 A from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin.
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chlorella virus Pyrimidine Dimer glycosylase and escherichia coli endonucleases iv and v have incision activity on 2 2 4 triamino 5 2h oxazolone
Genes and Environment, 2015Co-Authors: Katsuhito Kino, Shigenori Iwai, Masayo Suzuki, Masayuki Morikawa, Takanobu Kobayashi, Hiroshi MiyazawaAbstract:2,2,4-Triamino-5(2H)-oxazolone (Oz) in a DNA strand is an oxidation product of guanine and 8-oxo-7, 8-dihydroguanine, and such a lesion can cause G-to-C transversions. Previously, Fpg/Nei and Nth were shown to have incision activity on Oz. We investigated the activities of chlorella virus Pyrimidine Dimer glycosylase (cvPDG) and Escherichia coli endonucleases IV (Nfo) and V (Nfi) on Oz. Although the three enzymes have different repair mechanisms from Fpg/Nei and Nth, they still had incision activity on Oz. Given the incision activities of cvPDG, Nfo and Nfi on Oz in addition to Fpg/Nei and Nth, Oz is DNA damage that can be repaired by diverse enzymes.
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Flavin adenine dinucleotide chromophore charge controls the conformation of cyclobutane Pyrimidine Dimer photolyase α-helices.
Biochemistry, 2014Co-Authors: I Made Mahaputra Wijaya, Tatsuya Iwata, Junpei Yamamoto, Kenichi Hitomi, Shigenori Iwai, Elizabeth D. Getzoff, John T. M. Kennis, Tilo Mathes, Hideki KandoriAbstract:Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apoprotein, and substrate, so that only the initial, ultrafast, photon–chromophore reaction and the final, slow, protein conformational change provide separate, nonoverlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening time scales still cannot be fully deconvoluted. We overcome the problem by using a novel method to selectively isotope-label the apoprotein but not the flavin adenine dinucleotide (FAD) cofactor. This allowed the Fourier transform infrared (FTIR) signals to be separated from the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive structure–function study by FTIR spectroscopy of the Escherichia coli cyclobutane Pyrimidine Dimer photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair, which reveale...
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detection of distinct α helical rearrangements of cyclobutane Pyrimidine Dimer photolyase upon substrate binding by fourier transform infrared spectroscopy
Biochemistry, 2013Co-Authors: Made Mahaputra I Wijaya, Tatsuya Iwata, Junpei Yamamoto, Kenichi Hitomi, Shigenori Iwai, Elizabeth D. Getzoff, Yu Zhang, Hideki KandoriAbstract:Photolyases (PHRs) utilize near-ultraviolet (UV)–blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane Pyrimidine Dimer PHR (CPD-PHR) from Escherichia coli binds flavin adenine dinucleotide (FAD) as a cofactor and 5,10-methenyltetrahydrofolate as a light-harvesting pigment and specifically repairs CPD lesions. By comparison, a second photolyase known as (6–4) PHR, present in a range of higher organisms, uniquely repairs (6–4) PPs. To understand the repair mechanism and the substrate specificity that distinguish CPD-PHR from (6–4) PHR, we applied Fourier transform infrared (FTIR) spectroscopy to bacterial CPD-PHR in the presence or absence of a well-defined DNA substrate, as we have studied previously for vertebrate (6–4) PHR. PHRs show light-induced reduction of FAD, and photorepair by CPD-PHR involves the transfer of an electron from the photoexcited reduced FAD to the damaged DNA for cleaving the Dimers to maintain the DNA’s integrity. Here, we measur...
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photosensitized 2 2 cycloaddition of n acetylated cytosine affords stereoselective formation of cyclobutane Pyrimidine Dimer
Nucleic Acids Research, 2011Co-Authors: Junpei Yamamoto, Kosuke Nishiguchi, Koichiro Manabe, Chikahide Masutani, Fumio Hanaoka, Shigenori IwaiAbstract:Photocycloaddition between two adjacent bases in DNA produces a cyclobutane Pyrimidine Dimer (CPD), which is one of the major UV-induced DNA lesions, with either the cis-syn or trans-syn structure. In this study, we investigated the photosensitized intramolecular cycloaddition of partially-protected thymidylyl-(3 0 !5 0 )-N 4 -acetyl-2 0 deoxy-5-methylcytidine, to clarify the effect of the base modification on the cycloaddition reaction. The reaction resulted in the stereoselective formation of the trans-syn CPD, followed by hydrolysis of the acetylamino group. The same result was obtained for the photocycloaddition of thymidylyl(3 0 !5 0 )-N 4 -acetyl-2 0 -deoxycytidine, whereas both the cis-syn and trans-syn CPDs were formed from thymidylyl-(3 0 !5 0 )-thymidine. Kinetic analyses revealed that the activation energy of the acidcatalyzed hydrolysis is comparable to that reported for the thymine-cytosine CPD. These findings provided a new strategy for the synthesis of oligonucleotides containing the trans-syn CPD. Using the synthesized oligonucleotide, translesion synthesis by human DNA polymerase g was analyzed.
Amanda K Mccullough - One of the best experts on this subject based on the ideXlab platform.
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uncoupling of nucleotide flipping and dna bending by the t4 Pyrimidine Dimer dna glycosylase
Biochemistry, 2006Co-Authors: Randall K Walker, Amanda K Mccullough, Stephen R LloydAbstract:Bacteriophage T4 Pyrimidine Dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane Pyrimidine Dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5'-thymine of a thymine-thymine (TT) Dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60 degrees in the enzyme-substrate (ES) complex. To examine the kinetic details of the precatalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately assess nucleotide flipping and DNA bending. The fluorescent adenine base analogue, 2-aminopurine (2-AP), placed opposite an abasic site analogue, tetrahydrofuran, exhibited a 2.8-fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping, kon and koff pre-steady-state kinetic measurements were determined. DNA bending was assessed by fluorescence resonance energy transfer using fluorescent donor-acceptor pairs located at the 5'-ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5-fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.
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structure of t4 Pyrimidine Dimer glycosylase in a reduced imine covalent complex with abasic site containing dna
Journal of Molecular Biology, 2006Co-Authors: Gali Golan, Amanda K Mccullough, Stephen R Lloyd, M L Dodson, Dmitry O Zharkov, Arthur P Grollman, Gil ShohamAbstract:Abstract The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane Pyrimidine Dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 Pyrimidine Dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the α-amino group of the N-terminal threonine residue and the C1′ of the deoxyribose sugar of the 5′ Pyrimidine at the Dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA–protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing Pyrimidine Dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (∼ 66°) in the DNA at the Dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane Dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.
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chlorella virus Pyrimidine Dimer glycosylase excises ultraviolet radiation and hydroxyl radical induced products 4 6 diamino 5 formamidoPyrimidine and 2 6 diamino 4 hydroxy 5 formamidoPyrimidine from dna
Photochemistry and Photobiology, 2002Co-Authors: Pawel Jaruga, Ritche Jabil, Amanda K Mccullough, Henry Rodriguez, Miral Dizdaroglu, Stephen R LloydAbstract:A DNA glycosylase specific for UV radiation‐induced Pyrimidine Dimers has been identified from the Chlorella virus Paramecium Bursaria Chlorella virus-1. This enzyme (Chlorella virus Pyrimidine Dimer glycosylase [cvpdg]) exhibits a 41% amino acid identity with endonuclease V from bacteriophage T4 (T4 Pyrimidine Dimer glycosylase [T4-pdg]), which is also specific for Pyrimidine Dimers. However, cv-pdg possesses a higher catalytic efficiency and broader substrate specificity than T4-pdg. The latter excises 4,6-diamino-5-formamidoPyrimidine (FapyAde), a UV radiation‐ and hydroxyl radical‐induced monomeric product of adenine in DNA. Using gas chromatography‐isotope-dilution mass spectrometry and g-irradiated DNA, we show in this work that cv-pdg also displays a catalytic activity for excision of FapyAde and, in addition, it excises 2,6-diamino-4-hydroxy-5-formamidoPyrimidine (FapyGua). Kinetic data show that FapyAde is a better substrate for cv-pdg than FapyGua. On the other hand, cv-pdg possesses a greater efficiency
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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: Amanda K Mccullough, Matthew T Romberg, Simon G Nyaga, Yuanfen Wei, Thomas G Wood, Johnstephen Taylor, James L Van Etten, M L Dodson, Stephen R LloydAbstract:Abstract Endonuclease V from bacteriophage T4, is acis-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 β-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.
Eiko Ohtsuka - One of the best experts on this subject based on the ideXlab platform.
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DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane Pyrimidine Dimer
Nucleic Acids Research, 2000Co-Authors: Takuya Torizawa, Yasuo Komatsu, Eiko Ohtsuka, Tomonori Suzuki, Osamu Nikaido, Hiroshi Morioka, Nobuhiro Yamamoto, Kaoru Nobuoka, Koichi Kato, Ichio ShimadaAbstract:Monoclonal antibodies specific for the cyclobutane Pyrimidine Dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis-syncyclobutane thymine Dimer (T[c,s]T). 31 PN MR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[c,s]T) and d(TAT[c,s]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[c,s]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V H domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3′-side of the nucleotides onto the H1 and H3 segments, with the 5′-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.
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antigen structural requirements for recognition by a cyclobutane thymine Dimer specific monoclonal antibody
Nucleic Acids Research, 1997Co-Authors: Yasuo Komatsu, Tomomi Tsujino, Tomonori Suzuki, Osamu Nikaido, Eiko OhtsukaAbstract:A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane Pyrimidine Dimer (T[cis-syn]T) has previously been established; however,the immunization had used UV-irradiated calf-thymus DNA containing a heterogeneous mixture of photoproduct sites. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound with cis-syn,but not trans-syn thymine Dimer,and could bind strongly with four nucleotide analogs in which the cis-syn Pyrimidine Dimer was located in the center. Antigen analogs containing abasic linkers at the 5'- or 3'-side of the cis-syn cyclobutane Pyrimidine Dimer were synthesized and tested for binding to TDM-2. The results indicated that TDM-2 recognizes not only the cyclobutane ring but also both the 5'- and 3'-side nucleosides of the cyclobutane Dimer. Furthermore,it was proved that either the 5'- or 3'-side phosphate group at a cyclobutane Dimer site was absolutely required for the affinity to TDM-2. The antibody showed a strong binding to single stranded DNA but indicated little binding to double stranded DNA.
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atomic model of a Pyrimidine Dimer excision repair enzyme complexed with a dna substrate structural basis for damaged dna recognition
Cell, 1995Co-Authors: Dmitry G Vassylyev, Eiko Ohtsuka, Shigenori Iwai, Mariko Ariyoshi, Tatsuki Kashiwagi, Yuriko Mikami, Kosuke MorikawaAbstract:Abstract T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the Pyrimidine Dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine Dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60° inclination at the central thymine Dimer. The adenine base complementary to the 5′ side of the thymine Dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.
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crystal structure of a Pyrimidine Dimer specific excision repair enzyme from bacteriophage t4 refinement at 1 45 a and x ray analysis of the three active site mutants
Journal of Molecular Biology, 1994Co-Authors: Kosuke Morikawa, Katsuo Katayanagi, Mariko Ariyoshi, Dmitry G Vassylyev, Osamu Matsumoto, Eiko OhtsukaAbstract:Crystallographic study of bacteriophage T4 endonuclease V, which is involved in the initial step of the Pyrimidine Dimer-specific excision repair pathway, has been carried out with respect to the wild-type and three different mutant enzymes. This enzyme catalyzes the cleavage of the N -glycosyl bond at the 5′-side of the Pyrimidine Dimer, and subsequently incises the phosphodiester bond at the apyrimidinic site through a β-elimination reaction. The structure of the wild-type enzyme refined at 1.45 A resolution reveals the detailed molecular architecture. The enzyme is composed of a single compact domain classified as an all-α structure. The molecule is stabilized mainly by three hydrophobic cores, two of which include many aromatic side-chain interactions. The structure has a unique folding motif, where the amino-terminal segment penetrates between two major α-helices and prevents their direct contact, and it is incompatible with the close-packing category of helices for protein folding. The concave surface, covered with many positive charges, implies an interface for DNA binding. The glycosylase catalytic center, which comprises Glu23 and the surrounding basic residues Arg3, Arg22 and Arg26, lie in this basic surface. The crystal structures of the three active-site mutants, in which Glu23 was replaced by Gln(E23Q) and Asp (E23D), respectively, and Arg3 by Gln (R3Q), have been determined at atomic resolution. The backbone structures of the E23Q and R3Q mutants were almost identical with that of the wild-type, while the E23D mutation induces a small, but significant, change in the backbone structure, such as an increase of the central kink of the H1 helix at Pro25. In the catalytic center of the glycosylase, however, these three mutations do not generate notable movements of protein atoms, except for significant shifts of some bound water molecules. Thus, the structural differences between the wild-type and each mutant are confined to the remarkably small region around their replaced chemical groups. Combined with the biochemical studies and the difference circular dichroism measurements, these results allow us to conclude that the negatively charged carboxyl group of Glu23 is essential for the cleavage of the N -glycosyl bond, and that the positively charged guanidino group of Arg3 is crucial to bind the substrate, a DNA duplex containing a Pyrimidine Dimer. The amino terminal α-amino group is located at a position approximately 4.4 A away from the carboxyl group of Glu23. These structural features are generally consistent with the reaction scheme proposed by Dodson and co-workers.
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human nucleotide excision nuclease incises synthetic double stranded dna containing a Pyrimidine Dimer at the fourth phosphodiester linkage 3 to the Pyrimidine Dimer
Biochemistry, 1993Co-Authors: Satoshi Tateishi, Eiko Ohtsuka, Naoko Hori, Masaru YamaizumiAbstract:Linear 75mer double-stranded DNA containing a single Pyrimidine Dimer at a unique site was used to investigate Pyrimidine Dimer-dependent endonuclease activities from human cells. HeLaS3 cell extract incised the target DNA at the fourth phosphodiester linkage 3' to the Pyrimidine Dimer. However, incision of the DNA at 5' side of the Pyrimidine Dimer was not detected. The incision was also detected in cell extracts prepared from other excision repair-proficient cell lines. Incision was detected only on the DNA strand containing a Pyrimidine Dimer in the presence of poly(dI-dC)-poly(dI- dC) double strand. The reaction required Mg2+ but not ATP. The extract prepared from excision repair-deficient xeroderma pigmentosum (XP) cells belonging to the complementation group A was unable to incise the DNA. Extracts from the complementation groups C, D, and G incised the DNA very weakly at the third phosphodiester linkage 3' to the Pyrimidine Dimer, a site different from that incised by normal human cell extract. These results suggest that the observed incision reaction is associated with excision repair in human cells.
M L Dodson - One of the best experts on this subject based on the ideXlab platform.
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structure of t4 Pyrimidine Dimer glycosylase in a reduced imine covalent complex with abasic site containing dna
Journal of Molecular Biology, 2006Co-Authors: Gali Golan, Amanda K Mccullough, Stephen R Lloyd, M L Dodson, Dmitry O Zharkov, Arthur P Grollman, Gil ShohamAbstract:Abstract The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane Pyrimidine Dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 Pyrimidine Dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the α-amino group of the N-terminal threonine residue and the C1′ of the deoxyribose sugar of the 5′ Pyrimidine at the Dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA–protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing Pyrimidine Dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (∼ 66°) in the DNA at the Dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane Dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.
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role of his 16 in turnover of t4 Pyrimidine Dimer glycosylase
Journal of Biological Chemistry, 2004Co-Authors: Michael G Meador, Stephen R Lloyd, Lavanya Rajagopalan, M L DodsonAbstract:Abstract Previously, the histidine residue at position 16 in the mature T4 Pyrimidine Dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle.
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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: Amanda K Mccullough, Matthew T Romberg, Simon G Nyaga, Yuanfen Wei, Thomas G Wood, Johnstephen Taylor, James L Van Etten, M L Dodson, Stephen R LloydAbstract:Abstract Endonuclease V from bacteriophage T4, is acis-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 β-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.
