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Dmitry O. Zharkov - One of the best experts on this subject based on the ideXlab platform.
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Critical Sites of DNA Backbone Integrity for Damaged Base Removal by Formamidopyrimidine–DNA Glycosylase
Biochemistry, 2019Co-Authors: Anton V. Endutkin, Dmitry O. ZharkovAbstract:DNA Glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most Glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme’s active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by Formamidopyrimidine–DNA Glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3′ to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3′-terminal groups was more detrimental for t...
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critical sites of DNA backbone integrity for damaged base removal by Formamidopyrimidine DNA Glycosylase
Biochemistry, 2019Co-Authors: Anton V. Endutkin, Dmitry O. ZharkovAbstract:DNA Glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most Glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme’s active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by Formamidopyrimidine–DNA Glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3′ to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3′-terminal groups was more detrimental for t...
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residue coevolution reveals functionally important intramolecular interactions in Formamidopyrimidine DNA Glycosylase
DNA Repair, 2018Co-Authors: Anton V. Endutkin, Alexander V Popov, Simeon S Koptelov, Natalya A Torgasheva, Alexander A Lomzov, Alphiya R Tsygankova, Tatiana V Skiba, D A Afonnikov, Dmitry O. ZharkovAbstract:Abstract In protein evolution, functionally important intramolecular interactions, such as polar bridges or hydrophobic interfaces, tend to be conserved. We have analyzed coevolution of physicochemical properties in pairs of amino acid residues in the Formamidopyrimidine–DNA Glycosylase (Fpg) protein family, identified three conserved polar bridges (Arg54–Glu131, Gln234–Arg244, and Tyr170–Ser208 in the E. coli protein) located in known functional regions of the protein, and analyzed their roles by site-directed mutagenesis. The structure and molecular dynamic modeling showed that the coevolving pairs do not form isolated bridges but rather participate in tight local clusters of hydrogen bonds. The Arg54–Glu131 bridge, connecting the N- and C-terminal domains, was important for DNA binding, as its abolishment or even ion pair reversal inactivated Fpg and greatly decreased the enzyme’s affinity for DNA. Mutations of the Gln234–Arg244 bridge, located at the base of the single Fpg β-hairpin zinc finger, did not affect the activity but sharply decreased the melting temperature of the protein, with the bridge reversal partially restoring the thermal stability. Finally, Tyr170 mutation to Phe decreased Fpg binding but did not fully inactivate the protein, whereas Ser208 replacement with Ala had no effect; molecular dynamics showed that in both wild-type and S208 A Fpg, Tyr170 quickly re-orients to form an alternative set of hydrogen bonds. Thus, the coevolution analysis approach, combined with biochemical and computational studies, provides a powerful tool for understanding intramolecular interactions important for the function of DNA repair enzymes.
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molecular dynamics simulation of the opposite base preference and interactions in the active site of Formamidopyrimidine DNA Glycosylase
BMC Structural Biology, 2018Co-Authors: Alexander V Popov, Anton V. Endutkin, Yuri N Vorobjev, Dmitry O. ZharkovAbstract:Background Formamidopyrimidine-DNA Glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1′ of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg–DNA complexes have been solved, yet no structure with A opposite the lesion is available.
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Molecular dynamics simulation of the opposite-base preference and interactions in the active site of Formamidopyrimidine-DNA Glycosylase
BMC Structural Biology, 2017Co-Authors: Alexander V Popov, Anton V. Endutkin, Yuri N Vorobjev, Dmitry O. ZharkovAbstract:Background Formamidopyrimidine-DNA Glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1′ of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg–DNA complexes have been solved, yet no structure with A opposite the lesion is available. Results Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1–Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein–DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition. Conclusion Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.
Arthur P Grollman - One of the best experts on this subject based on the ideXlab platform.
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a dynamic checkpoint in oxidative lesion discrimination by Formamidopyrimidine DNA Glycosylase
Nucleic Acids Research, 2016Co-Authors: Haoquan Li, Dmitry O. Zharkov, Anton V. Endutkin, Arthur P Grollman, Christina Bergonzo, Arthur J Campbell, Carlos De Los Santos, Carlos SimmerlingAbstract:In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme-DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, Formamidopyrimidine-DNA Glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson-Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes.
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substrate discrimination by Formamidopyrimidine DNA Glycosylase a mutational analysis
Journal of Biological Chemistry, 2004Co-Authors: Elena Zaika, R Gilboa, E Matz, Arthur P Grollman, Rebecca A Perlow, Suse Broyde, Dmitry O. ZharkovAbstract:Abstract Formamidopyrimidine-DNA Glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.
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structural characterization of the fpg family of DNA Glycosylases
DNA Repair, 2003Co-Authors: Dmitry O. Zharkov, G Shoham, Arthur P GrollmanAbstract:Abstract Until recently, the Fpg family was the only major group of DNA Glycosylases for which no structural data existed. Prototypical members of this family, found in eukaryotes as well as prokaryotes, have now been crystallized as free proteins and as complexes with DNA. In this review, we analyze the available structural information for Formamidopyrimidine-DNA Glycosylase (Fpg) and endonuclease VIII (Nei). Special emphasis is placed on mechanisms by which these enzymes recognize and selectively excise cognate lesions from oxidatively damaged DNA. The problem of lesion recognition is considered in two parts: how the enzyme efficiently locates a single lesion embedded in a vast excess of DNA; and how the lesion is accommodated in a pocket near the active site of the enzyme. Although all crystal structures reported to date for the Fpg family lack the damaged base, functionally important residues that participate in DNA binding and enzyme catalysis have been clearly identified and other residues, responsible for substrate specificity, have been inferred.
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structure of Formamidopyrimidine DNA Glycosylase covalently complexed to DNA
Journal of Biological Chemistry, 2002Co-Authors: R Gilboa, Dmitry O. Zharkov, G Golan, Andrea Fernandes, Sue Ellen Gerchman, E Matz, Jadwiga H Kycia, Arthur P Grollman, G ShohamAbstract:Abstract Formamidopyrimidine-DNA Glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction betweenEscherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.
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nh2 terminal proline acts as a nucleophile in the Glycosylase ap lyase reaction catalyzed by escherichia coli Formamidopyrimidine DNA Glycosylase fpg protein
Journal of Biological Chemistry, 1997Co-Authors: Dmitry O. Zharkov, Robert Rieger, Charles R Iden, Arthur P GrollmanAbstract:Abstract Formamidopyrimidine-DNA Glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA Glycosylase), β-elimination (AP lyase), and δ-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.
Serge Boiteux - One of the best experts on this subject based on the ideXlab platform.
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structural basis for the recognition of the fapydg lesion 2 6 diamino 4 hydroxy 5 Formamidopyrimidine by Formamidopyrimidine DNA Glycosylase
Journal of Biological Chemistry, 2004Co-Authors: Franck Coste, Serge Boiteux, C Zelwer, Matthias Ober, Thomas Carell, Bertrand CastaingAbstract:Abstract Formamidopyrimidine-DNA Glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-Formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity.
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analysis of 8 hydroxyguanine 8 oh gua released from DNA by the Formamidopyrimidine DNA Glycosylase fpg protein a reliable method to estimate cellular oxidative stress
Journal of Radiation Research, 2004Co-Authors: Hiroshi Orimo, Serge Boiteux, Yoshiki Tokura, Hiroshi KasaiAbstract:8-OH-Gua background level/Fpg protein/γ-irradiation. To improve the analyses of a form of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), we treated isolated DNA with Formamidopyrimidine DNA Glycosylase (Fpg) and analyzed the released 8-OH-Gua by using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The human lung carcinoma cells (A549) and human keratinocyte (HaCaT) were irradiated with γ-rays. After the isolated DNA was treated with the Fpg protein, we analyzed the released 8-OH-Gua by using an HPLC-ECD. With this method, the background level of 8-OH-Gua in DNA from human lung carcinoma cells was determined to be 3.4 residues per 10 7 guanine (Gua). A similar background level of 8-OH-Gua (3.1 residues per 10 7 Gua) was also detected in human keratinocyte DNA with this method. These background 8-OH-Gua levels in cellular DNA are comparable to that obtained previously by an analysis of 8-OH-dGMP after nuclease P1 digestion of cellular DNA (4.3 residues per 10 7 dCMP). A dose-dependent increase of 8-OH-Gua (0.17/10 7 Gua/Gy) was observed after cells were irradiated with γ-rays. Twenty hours after γ-irradiation with 60 Gy, 75% of the 8-OH-Gua produced in keratinocyte DNA was repaired. With our new analysis method, it is possible to detect the small changes in the 8-OH-Gua levels in cellular DNA induced by various environmental factors.
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crystal structure of the lactococcus lactis Formamidopyrimidine DNA Glycosylase bound to an abasic site analogue containing DNA
The EMBO Journal, 2002Co-Authors: Laurence Serre, Serge Boiteux, Karine Pereira De Jesus, C Zelwer, Bertrand CastaingAbstract:The Formamidopyrimidine-DNA Glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA Glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60° bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.
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crystal structure of the lactococcus lactis Formamidopyrimidine DNA Glycosylase bound to an abasic site analogue containing DNA
The EMBO Journal, 2002Co-Authors: Laurence Serre, Serge Boiteux, Karine Pereira De Jesus, C Zelwer, Bertrand CastaingAbstract:The Formamidopyrimidine-DNA Glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA Glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8-oxoguanine and imidazole ring-opened purines, from oxidatively damaged DNA. The structure of a non-covalent complex between the Lactoccocus lactis Fpg and a 1,3-propanediol (Pr) abasic site analogue-containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60 degrees bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8-oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.
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Formamidopyrimidine DNA Glycosylase in the yeast Saccharomyces cerevisiae
Nucleic Acids Research, 1994Co-Authors: Regina De Oliveira, Patricia Auffret Van Der Kemp, Albert Geiger, Peter Nehls, Dominique Thomas, Serge BoiteuxAbstract:A DNA Glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylFormamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA Glycosylase has a molecular weight of about 40 kDa. The Fapy DNA Glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA Glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA Glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.
R Santus - One of the best experts on this subject based on the ideXlab platform.
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effect of single mutations on the structural dynamics of a DNA repair enzyme the escherichia coli Formamidopyrimidine DNA Glycosylase a fluorescence study using tryptophan residues as reporter groups
FEBS Journal, 1998Co-Authors: S V Kuznetsov, Jacques Laval, Olga M Sidorkina, Juan Jurado, M Bazin, Patrick Tauc, Jeanclaude Brochon, R SantusAbstract:The effects on the structure dynamics of the Escherichia coli wild-type Formamidopyrimidine-DNA Glycosylase (Fpg) protein of the single mutations Lys57!Gly (FpgK57G), Pro2!Gly (FpgP2G) and Pro2!Glu (FpgP2E) were studied by fluorescence techniques, namely: lifetime measurements and acrylamide quenching of the fluorescence of Trp residues. The fluorescence decays of Fpg and its mutant forms were analysed by the maximum-entropy method and lifetime distributions in the range 200 ps to 9 ns were obtained. The lifetime distribution profiles of FpgK57G, FpgP2G and FpgP2E are different from that of wild-type Fpg. Both dynamic and static quenching by acrylamide were observed for all the proteins. At 20 °C, the bimolecular collisional quenching rate constant of the FpgP2E fluorescence by acrylamide was only 0.8 M 21 s 21 as compared to about 1.4 M 21 s 21 for the three other proteins. At 6 °C, all the spectroscopic properties of these four proteins are about the same. The analysis of experimental data demonstrates that all three mutations induce a structural reorganization of the Fpg protein. However, only the P2E mutation lead to a reduced accessibility of some Trp residues to acrylamide quenching. It is concluded that the single P2E replacement induces a conformational change leading to a more rigid globular structure as opposed to the wild type and K57G and P2G mutations. The influence of the single mutations on the enzyme activities of the Fpg protein is discussed.
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effect of single mutations on the structural dynamics of a DNA repair enzyme the escherichia coli Formamidopyrimidine DNA Glycosylase a fluorescence study using tryptophan residues as reporter groups
FEBS Journal, 1998Co-Authors: S V Kuznetsov, Jacques Laval, Olga M Sidorkina, Juan Jurado, M Bazin, Patrick Tauc, Jeanclaude Brochon, R SantusAbstract:: The effects on the structure dynamics of the Escherichia coli wild-type Formamidopyrimidine-DNA Glycosylase (Fpg) protein of the single mutations Lys57-->Gly (FpgK57G), Pro2-->Gly (FpgP2G) and Pro2-->Glu (FpgP2E) were studied by fluorescence techniques, namely: lifetime measurements and acrylamide quenching of the fluorescence of Trp residues. The fluorescence decays of Fpg and its mutant forms were analysed by the maximum-entropy method and lifetime distributions in the range 200 ps to 9 ns were obtained. The lifetime distribution profiles of FpgK57G, FpgP2G and FpgP2E are different from that of wild-type Fpg. Both dynamic and static quenching by acrylamide were observed for all the proteins. At 20 degrees C, the bimolecular collisional quenching rate constant of the FpgP2E fluorescence by acrylamide was only 0.8 M(-1) s(-1) as compared to about 1.4 M(-1) s(-1) for the three other proteins. At 6 degrees C, all the spectroscopic properties of these four proteins are about the same. The analysis of experimental data demonstrates that all three mutations induce a structural reorganization of the Fpg protein. However, only the P2E mutation lead to a reduced accessibility of some Trp residues to acrylamide quenching. It is concluded that the single P2E replacement induces a conformational change leading to a more rigid globular structure as opposed to the wild type and K57G and P2G mutations. The influence of the single mutations on the enzyme activities of the Fpg protein is discussed.
Guido Frosina - One of the best experts on this subject based on the ideXlab platform.
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accelerated repair and reduced mutagenicity of DNA damage induced by cigarette smoke in human bronchial cells transfected with e coli Formamidopyrimidine DNA Glycosylase
PLOS ONE, 2014Co-Authors: Mara Foresta, Alessandro Poggi, Alberto Izzotti, Sebastiano La Maestra, Rosanna T Micale, Donatella Vecchio, Guido FrosinaAbstract:Cigarette smoke (CS) is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH) inducing DNA damage. The bacterial DNA repair enzyme Formamidopyrimidine DNA Glycosylase (FPG) repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP). Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na+K+-ATPase locus (ouar) were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells’ capacity to repair damaged DNA.
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Increased resistance to oxidative DNA damage of trabecular meshwork cells by E. coli FPG gene transfection.
Free Radical Research, 2011Co-Authors: Mara Foresta, Guido Frosina, Sergio Claudio Saccà, Cristina Cartiglia, Mariagrazia Longobardi, Alberto IzzottiAbstract:AbstractOxidative damage plays a pathogenic role in various chronic degenerative diseases. Oxidative damage targeting trabecular meshwork (TM) cells as a consequence of mitochondrial damage is a pathogenic mechanism for glaucoma, the most common cause of irreversible blindness worldwide. Consequences of oxidative damage are attenuated by endocellular activities involved in scavenging reactive oxidative species and DNA repair. Selected bacterial genes are highly efficient at protecting cells from oxidative DNA damage. This situation occurs for Escherichia coli Formamidopyrimidine DNA Glycosylase (FPG), a major DNA Glycosylase that repairs oxidatively damaged DNA. Accordingly, this study was aimed at transfecting human TM cells (HTMC) with Fpg in order to increase their resistance to oxidative damage. This study demonstrates that it is feasible to increase resistance of HTMC to endogenous oxidative damage by gene transfection. These findings bear relevance for primary and secondary prevention of degenerativ...
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defective repair of 5 hydroxy 2 deoxycytidine in cockayne syndrome cells and its complementation by escherichia coli Formamidopyrimidine DNA Glycosylase and endonuclease iii
Free Radical Biology and Medicine, 2010Co-Authors: Mara Foresta, Monica Ropolo, Paolo Degan, Alessandro Poggi, Ilaria Pettinati, Gianluca Damonte, Guido FrosinaAbstract:Abstract Repair of the oxidized purine 8-oxo-7,8-dihydro-2′-deoxyguanosine is inefficient in cells belonging to both complementation groups A and B of Cockayne syndrome (CS), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that both CS-A and CS-B cells are also defective in the repair of 5-hydroxy-2′-deoxycytidine (5-OHdC), an oxidized pyrimidine with cytotoxic and mutagenic properties. The defect in the repair of oxidatively damaged DNA in CS cells thus extends to oxidized pyrimidines, indicating a general flaw in the repair of oxidized lesions in this syndrome. The defect could not be reproduced in in vitro repair experiments on oligonucleotide substrates, suggesting a role for both CS-A and CS-B proteins in chromatin remodeling during 5-OHdC repair. Expression of Escherichia coli Formamidopyrimidine DNA Glycosylase (FPG) or endonuclease III complemented the 5-OHdC repair deficiency. Hence, the expression of a single enzyme, FPG from E. coli, stably corrects the delayed removal of both oxidized purines and oxidized pyrimidines in CS cells.
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complementation of the oxidatively damaged DNA repair defect in cockayne syndrome a and b cells by escherichia coli Formamidopyrimidine DNA Glycosylase
Free Radical Biology and Medicine, 2007Co-Authors: Monica Ropolo, Paolo Degan, Mara Foresta, Mariarosaria Derrico, Denise Lasiglie, Eugenia Dogliotti, Gianluigi Casartelli, Simonetta Zupo, Alessandro Poggi, Guido FrosinaAbstract:Repair of the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoGua) is inefficient in cells belonging to the B complementation group of Cockayne syndrome (CS-B), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that cells belonging to the A complementation group (CS-A) are also defective in repair of 8-oxoGua and we demonstrate that expression of the Escherichia coli Formamidopyrimidine DNA Glycosylase (FPG) completely corrects the repair deficiency in both CS-A and CS-B cells. Phenotypically, CS-A cells are normally sensitive to toxicity and micronuclei induced by the oxidizing agent potassium bromate. CS-B cells display sensitivity to elevated concentrations of potassium bromate but this is not compensated by FPG expression, suggesting toxicity of lesions that are not FPG substrates. The data indicate that 8-oxoGua is not a major toxic and clastogenic lesion in CS cells.
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prophylaxis of oxidative DNA damage by Formamidopyrimidine DNA Glycosylase
International Journal of Cancer, 2006Co-Authors: Guido FrosinaAbstract:Lying at the gas-exchange interface, lung epithelia may be at risk of oxidation-induced mutagenesis. Further, inflammation processes possibly consequent on smoking liberate reactive oxygen species that multiply the carcinogenic effects of tobacco. DNA repair mechanisms play a major role in counteracting the deleterious effects of oxidative DNA damage. Some studies find positive associations between lung cancer and variations in the human 8-oxoguanine DNA Glycosylase (hOGG1) gene that encodes a major DNA Glycosylase for oxidized lesions with sluggish kinetics properties. The bacterial homologue Formamidopyrimidine-DNA Glycosylase (FPG) is 80-fold faster than hOGG1 in repairing mutagenic oxidative lesions. Cell-culture studies have shown that FPG can be expressed in mammalian cells, where it accelerates DNA repair and abates mutagenicity of a wide range of DNA-damaging agents. Prophylaxis of oxidative DNA damage and mutation could be achieved in lung epithelia and other tissues of at-risk individuals by expression of the FPG protein. Currently available vehicles for this peculiar type of gene therapy are briefly surveyed. © 2006 Wiley-Liss, Inc.