The Experts below are selected from a list of 20718 Experts worldwide ranked by ideXlab platform
Alexander C Drohat - One of the best experts on this subject based on the ideXlab platform.
-
structural basis for excision of 5 formylcytosine by thymine dna glycosylase
Biochemistry, 2016Co-Authors: Lakshmi Swarna Mukhi Pidugu, Joshua W Flowers, Christopher T Coey, Marc M Greenberg, Edwin Pozharski, Alexander C DrohatAbstract:Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epigenetic regulation. Performing a critical step in a pathway for active DNA demethylation, TDG removes 5-formylcytosine and 5-carboxylcytosine, oxidized derivatives of 5-methylcytosine that are generated by TET (ten–eleven translocation) enzymes. We determined a crystal structure of TDG bound to DNA with a noncleavable (2′-fluoroarabino) analogue of 5-formyldeoxycytidine flipped into its active site, revealing how it recognizes and hydrolytically Excises fC. Together with previous structural and biochemical findings, the results illustrate how TDG employs an adaptable active site to Excise a broad variety of nucleobases from DNA.
-
thymine dna glycosylase exhibits negligible affinity for nucleobases that it removes from dna
Nucleic Acids Research, 2015Co-Authors: Shuja Shafi Malik, Alexander C Drohat, Christopher T Coey, Kristen M Varney, Edwin PozharskiAbstract:: Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G ·: T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG Excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten-eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 A) structures of TDG enzyme-product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme-substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the Excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the Excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the Excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme-product complex.
Murat Saparbaev - One of the best experts on this subject based on the ideXlab platform.
-
The Human Oxidative DNA Glycosylase NEIL1 Excises Psoralen-induced Interstrand DNA Cross-links in a Three-stranded DNA Structure
The Journal of biological chemistry, 2009Co-Authors: Sophie Couvé, Gaëtane Macé-aimé, Filippo Rosselli, Murat SaparbaevAbstract:Previously, we have demonstrated that human oxidative DNA glycosylase NEIL1 Excises photoactivated psoralen-induced monoadducts but not genuine interstrand cross-links (ICLs) in duplex DNA. It has been postulated that the repair of ICLs in mammalian cells is mainly linked to DNA replication and proceeds via dual incisions in one DNA strand that bracket the cross-linked site. This process, known as "unhooking," enables strand separation and translesion DNA synthesis through the gap, yielding a three-stranded DNA repair intermediate composed of a short unhooked oligomer covalently bound to the duplex. At present, the detailed molecular mechanism of ICL repair in mammalian cells remains unclear. Here, we constructed and characterized three-stranded DNA structures containing a single ICL as substrates for the base excision repair proteins. We show that NEIL1 Excises with high efficiency the unhooked ICL fragment within a three-stranded DNA structure. Complete reconstitution of the repair of unhooked ICL shows that it can be processed in a short patch base excision repair pathway. The new substrate specificity of NEIL1 points to a preferential involvement in the replication-associated repair of ICLs. Based on these data, we propose a model for the mechanism of ICL repair in mammalian cells that implicates the DNA glycosylase activity of NEIL1 downstream of Xeroderma Pigmentosum group F/Excision Repair Cross-Complementing 1 endonuclease complex (XPF/ERCC1) and translesion DNA synthesis repair steps. Finally, our data demonstrate that Nei-like proteins from Escherichia coli to human cells can Excise bulky unhooked psoralen-induced ICLs via hydrolysis of glycosidic bond between cross-linked base and deoxyribose sugar, thus providing an alternative heuristic solution for the removal of complex DNA lesions.
-
substrate specificity of homogeneous monkeypox virus uracil dna glycosylase
Biochemistry, 2007Co-Authors: Sophie Duraffour, Murat Saparbaev, Alexander A Ishchenko, Jeanmarc Crance, Daniel GarinAbstract:Weak or nonexistent smallpox immunity in today's human population raises concerns about the possibility of natural or provoked genetic modifications leading to re-emergence of variola virus and other poxviruses. Thus, the development of new antiviral strategies aimed at poxvirus infections in humans is a high priority. The DNA repair protein uracil-DNA glycosylase (UNG) is one of the viral enzymes important for poxvirus pathogenesis. Consequently, the inhibition of UNG is a rational therapeutic strategy for infections with poxviruses. Monkeypox virus, which occurs naturally in Africa, can cause a smallpox-like disease in humans. Here, the monkeypox virus UNG (mpUNG) is characterized and compared to vaccinia virus UNG (vUNG) and human UNG (hUNG). The mpUNG protein Excises uracil preferentially from single-stranded DNA. Furthermore, mpUNG prefers the U·G pair over the U·A pair and does not Excise oxidized bases. Both mpUNG and vUNG viral proteins are strongly inhibited by physiological concentrations of NaC...
-
escherichia coli saccharomyces cerevisiae rat and human 3 methyladenine dna glycosylases repair 1 n6 ethenoadenine when present in dna
Nucleic Acids Research, 1995Co-Authors: Murat Saparbaev, Karol Kleibl, Jacques LavalAbstract:Abstract The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which generate various ethenobases in DNA. It has been reported that 1,N6-ethenoadenine (epsilon A) is Excised by a DNA glycosylase present in human cell extracts, whereas protein extracts from Escherichia coli and yeast were devoid of such an activity. We confirm that the human 3-methyladenine-DNA glycosylase (ANPG protein) Excises epsilon A residues. This finding was extended to the rat (ADPG protein). We show, at variance with the previous report, that pure E.coli 3-methyladenine-DNA glycosylase II (AlkA protein) as well as its yeast counterpart, the MAG protein, Excise epsilon A from double stranded oligodeoxynucleotides that contain a single epsilon A. Both enzymes act as DNA glycosylases. The full length and the truncated human (ANPG 70 and 40 proteins, respectively) and the rat (ADPG protein) 3-methyladenine-DNA glycosylases activities towards epsilon A are 2-3 orders of magnitude more efficient than the E.coli or yeast enzyme for the removal of epsilon A. The Km of the various proteins were measured. They are 24, 200 and 800 nM for the ANPG, MAG and AlkA proteins respectively. These three proteins efficiently cleave duplex oligonucleotides containing epsilon A positioned opposite T, G, C or epsilon A. However the MAG protein Excises A opposite cytosine much faster than opposite thymine, guanine or adenine.
Dmitry O. Zharkov - One of the best experts on this subject based on the ideXlab platform.
-
correlated cleavage of damaged dna by bacterial and human 8 oxoguanine dna glycosylases
Biochemistry, 2008Co-Authors: Viktoriya S Sidorenko, Dmitry O. ZharkovAbstract:Many enzymes acting on specific rare lesions in DNA are suggested to search for their targets by facilitated one-dimensional diffusion. We have used a recently developed correlated cleavage assay to investigate whether this mechanism operates for Fpg and OGG1, two structurally unrelated DNA glycosylases that Excise an important oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), from DNA. Similar to a number of other DNA glycosylases or restriction endonucleases, Fpg and OGG1 processively Excised 8-oxoG from pairs with cytosine at low salt concentrations, indicating that the lesion search likely proceeds by one-dimensional diffusion. At high salt concentrations, both enzymes switched to a distributive mode of lesion search. Correlated cleavage of abasic site-containing substrates proceeded in the same manner as cleavage of 8-oxoG. Interestingly, both Fpg and especially OGG1 demonstrated higher processivity if the substrate contained 8-oxoG·A pairs, against which these enzyme discriminate. Introduction of...
-
NEIL1 Excises 3' end proximal oxidative DNA lesions resistant to cleavage by NTH1 and OGG1.
Nucleic acids research, 2005Co-Authors: Jason L. Parsons, Dmitry O. Zharkov, Grigory L. DianovAbstract:Base excision repair is the major pathway for the repair of oxidative DNA damage in human cells that is initiated by a damage-specific DNA glycosylase. In human cells, the major DNA glycosylases for the excision of oxidative base damage are OGG1 and NTH1 that Excise 8-oxoguanine and oxidative pyrimidines, respectively. We find that both enzymes have limited activity on DNA lesions located in the vicinity of the 3' end of a DNA single-strand break, suggesting that other enzymes are involved in the processing of such lesions. In this study, we identify and characterize NEIL1 as a major DNA glycosylase that Excises oxidative base damage located in close proximity to the 3' end of a DNA single-strand break.
-
substrate discrimination by formamidopyrimidine dna glycosylase distinguishing interactions within the active site
Biochemistry, 2004Co-Authors: Rebecca A Perlowpoehnelt, And Arthur P Grollman, Dmitry O. Zharkov, Suse BroydeAbstract:Reactive oxygen species are byproducts of normal aerobic respiration and ionizing radiation, and they readily react with DNA to form a number of base lesions, including the mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5-formamidopyrimidine (FapyA), and 8-oxo-7,8-dihydroadenine (8-oxoA). Such oxidative lesions are removed by the base excision repair pathway, which is initiated by DNA glycosylases such as the formamidopyrimidine-DNA glycosylase (Fpg) in Escherichia coli. The 8-oxoG, FapyG, and FapyA lesions are bound and Excised by Fpg, while structurally similar 8-oxoA is Excised by Fpg very poorly. We carried out molecular modeling and molecular dynamics simulations to interpret substrate discrimination within the active site of E. coli Fpg. Lys-217 and Met-73 were identified as residues playing important roles in the recognition of the oxidized imidazole ring in the substrate bases, and the Watson-Crick edge of the damaged base plays a role in optimally positioning the base within the active site. The recognition and excision of FapyA likely result from the opened imidazole ring, while 8-oxoA's lack of flexibility and closed imidazole ring may contribute to Fpg's inability to Excise this base. Different interactions between each base and the enzyme specificity pocket account for differential treatment of the various lesions by this enzyme, and thus elucidate the structure-function relationship involved in an initial step of base excision repair.
Edwin Pozharski - One of the best experts on this subject based on the ideXlab platform.
-
structural basis for excision of 5 formylcytosine by thymine dna glycosylase
Biochemistry, 2016Co-Authors: Lakshmi Swarna Mukhi Pidugu, Joshua W Flowers, Christopher T Coey, Marc M Greenberg, Edwin Pozharski, Alexander C DrohatAbstract:Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epigenetic regulation. Performing a critical step in a pathway for active DNA demethylation, TDG removes 5-formylcytosine and 5-carboxylcytosine, oxidized derivatives of 5-methylcytosine that are generated by TET (ten–eleven translocation) enzymes. We determined a crystal structure of TDG bound to DNA with a noncleavable (2′-fluoroarabino) analogue of 5-formyldeoxycytidine flipped into its active site, revealing how it recognizes and hydrolytically Excises fC. Together with previous structural and biochemical findings, the results illustrate how TDG employs an adaptable active site to Excise a broad variety of nucleobases from DNA.
-
thymine dna glycosylase exhibits negligible affinity for nucleobases that it removes from dna
Nucleic Acids Research, 2015Co-Authors: Shuja Shafi Malik, Alexander C Drohat, Christopher T Coey, Kristen M Varney, Edwin PozharskiAbstract:: Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G ·: T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG Excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten-eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 A) structures of TDG enzyme-product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme-substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the Excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the Excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the Excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme-product complex.
Chongsoon Lee - One of the best experts on this subject based on the ideXlab platform.
-
excision repair of 2 5 diaziridinyl 1 4 benzoquinone dzq dna adduct by bacterial and mammalian 3 methyladenine dna glycosylases
Molecules and Cells, 2000Co-Authors: Chongsoon LeeAbstract:The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to Excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formami-dopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme Excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, Excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.
