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Tom Ellenberger - One of the best experts on this subject based on the ideXlab platform.
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molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase aag
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Michael D. Wyatt, Brian J Glassner, Leona D Samson, Tom EllenbergerAbstract:The human 3-methyladenine DNA glycosylase [alkyladenine DNA glycosylase (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other DNA Glycosylases that are specific for a particular type of damaged base, AAG excises a chemically diverse selection of substrate bases damaged by alkylation or deamination. The 2.1-Å crystal structure of AAG complexed to DNA containing 1,N6-ethenoadenine suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base in the crystal structures suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG.
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Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase.
Mutation Research-dna Repair, 2000Co-Authors: T Hollis, Tom EllenbergerAbstract:Abstract Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair Glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA–DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a SN1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.
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dna bending and a flip out mechanism for base excision by the helix hairpin helix dna glycosylase escherichia coli alka
The EMBO Journal, 2000Co-Authors: T Hollis, Y. Ichikawa, Tom EllenbergerAbstract:The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an SN1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH Glycosylases can bind to DNA in a similar manner.
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DNA bending and a flip-out mechanism for base excision by the helix- hairpin-helix DNA glycosylase, Escherichia coli AlkA
Embo J, 2000Co-Authors: T Hollis, Y. Ichikawa, Tom EllenbergerAbstract:The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin- helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH Glycosylases can bind to DNA in a similar manner.
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DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA
The EMBO Journal, 2000Co-Authors: T Hollis, Y. Ichikawa, Tom EllenbergerAbstract:The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH Glycosylases can bind to DNA in a similar manner.
Jacques Laval - One of the best experts on this subject based on the ideXlab platform.
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the type of dna glycosylase determines the base excision repair pathway in mammalian cells
Journal of Biological Chemistry, 1999Co-Authors: Paola Fortini, Eleonora Parlanti, Olga M Sidorkina, Jacques Laval, Eugenia DogliottiAbstract:Abstract The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA Glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N 6-ethenoadenine (eA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/β-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, eA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and eA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase β-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template.
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specific binding of a designed pyrrolidine abasic site analog to multiple dna Glycosylases
Journal of Biological Chemistry, 1998Co-Authors: Orlando D Scharer, Josef Jiricny, Jacques Laval, Huw M Nash, Gregory L VerdineAbstract:Abstract In the base excision DNA repair pathway, DNA Glycosylases recognize damaged bases in DNA and catalyze their excision through hydrolysis of the N-glycosidic bond. Attempts to understand the structural basis for DNA damage recognition by DNA Glycosylases have been hampered by the short-lived association of these enzymes with their DNA substrates. To overcome this problem, we have employed an approach involving the design and synthesis of inhibitors that form stable complexes with DNA Glycosylases, which can then be studied biochemically and structurally. We have previously reported that double-stranded DNA containing a pyrrolidine abasic site analog (PYR) forms an extremely stable complex with the DNA glycosylase AlkA and potently inhibits the reaction catalyzed by the enzyme (Scharer, O. D., Ortholand, J.-Y., Ganesan, A., Ezaz-Nikpay, K., and Verdine, G. L. (1995) J. Am. Chem. Soc.117, 6623–6624). Here we investigate the interaction of this inhibitor with a variety of additional DNA Glycosylases. With the exception of uracil DNA glycosylase all the Glycosylases tested bind specifically to PYR-containing oligonucleotides. By comparing the interaction of DNA Glycosylases with PYR and the structurally related tetrahydrofuran abasic site analog, we assess the importance of the positively charged ammonium group of the pyrrolidine in binding to the active site of these enzymes. Such a general inhibitor of DNA glycosyases provides a valuable tool to study stable complexes of these enzymes bound to substrate-like molecules.
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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.
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excision of hypoxanthine from dna containing dimp residues by the escherichia coli yeast rat and human alkylpurine dna Glycosylases
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Murat Saparbaev, Jacques LavalAbstract:Abstract The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA Glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested.
Chunyang Zhang - One of the best experts on this subject based on the ideXlab platform.
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integration of single molecule detection with magnetic separation for multiplexed detection of dna Glycosylases
Chemical Communications, 2018Co-Authors: Chenchen Li, Yan Zhang, Bo Tang, Chunyang ZhangAbstract:We combine single-molecule detection with magnetic separation for simultaneous measurement of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycosylase (UDG) based on excision repair-initiated endonuclease IV (Endo IV)-assisted signal amplification. This method can sensitively detect multiple DNA Glycosylases, and it can be further applied for the simultaneous measurement of enzyme kinetic parameters and screening of both hOGG1 and UDG inhibitors.
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simultaneous sensitive detection of multiple dna Glycosylases from lung cancer cells at the single molecule level
Chemical Science, 2018Co-Authors: Juan Hu, Bo Tang, Ying Li, Chunyang ZhangAbstract:DNA Glycosylases are involved in the base excision repair pathway, and all mammals express multiple DNA Glycosylases to maintain genome stability. However, the simultaneous detection of multiple DNA glycosylase still remains a great challenge. Here, we develop a single-molecule detection method for the simultaneous detection of human 8-oxoguanine DNA glycosylase 1 (hOGG1) and human alkyladenine DNA glycosylase (hAAG) on the basis of DNA glycosylase-mediated cleavage of molecular beacons. We designed a Cy3-labeled molecular beacon modified with 8-oxoguanine (8-oxoG) for a hOGG1 assay and a Cy5-labeled molecular beacon modified with deoxyinosine for a hAAG assay. hOGG1 may catalyze the removal of 8-oxoG from 8-oxoG/C base pairs to generate an apurinic/apyrimidinic (AP) site, and hAAG may catalyze the removal of deoxyinosine from deoxyinosine/T base pairs to generate an AP site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of AP sites results in the cleavage of molecular beacons, with Cy3 indicating the presence of hOGG1 and Cy5 indicating the presence of hAAG. Both of the Cy3 and Cy5 signals can be simply quantified by total internal reflection fluorescence-based single-molecule detection. This method can simultaneously detect multiple DNA Glycosylases with a detection limit of 2.23 × 10−6 U μL−1 for hOGG1 and 8.69 × 10−7 U μL−1 for hAAG without the involvement of any target amplification. Moreover, this method can be used for the screening of enzyme inhibitors and the simultaneous detection of hOGG1 and hAAG from lung cancer cells, having great potential for further application in early clinical diagnosis.
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homogeneously sensitive detection of multiple dna Glycosylases with intrinsically fluorescent nucleotides
Analytical Chemistry, 2017Co-Authors: Yan Zhang, Chenchen Li, Bo Tang, Chunyang ZhangAbstract:DNA Glycosylases are responsible for recognition and excision of the damaged bases in the base excision repair pathway, and all mammals express multiple DNA Glycosylases to maintain genome stability. However, simultaneous detection of multiple DNA glycosylase still remains a great challenge. Here, we develop a rapid and sensitive fluorescent method for simultaneous detection of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycolase (UDG) using exonuclease-assisted recycling signal amplification in combination with fluorescent bases 2-aminopurine (2-AP) and pyrrolo-dC (P-dC) as the fluorophores. We design a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with the trigger probes to form a sandwiched DNA substrate for hOGG1 and UDG. In addition, we design 2-AP and P-dC signal probes as the hairpin structures with 2-AP and P-dC in the stems. The presence of hOGG1 and UDG may initiate the signal amplification process by the recycling lambda exonuclease digest...
Murat Saparbaev - One of the best experts on this subject based on the ideXlab platform.
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pre steady state fluorescence analysis of damaged dna transfer from human dna Glycosylases to ap endonuclease ape1
Biochimica et Biophysica Acta, 2014Co-Authors: Alexandra A Kuznetsova, Nikita A Kuznetsov, Alexander A Ishchenko, Murat Saparbaev, Olga S FedorovaAbstract:Abstract Background DNA Glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Methods Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA Glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA Glycosylases AAG, OGG1, NEIL1, MBD4 cat and UNG from different structural superfamilies were used. Results We found that all DNA Glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. Conclusions We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA Glycosylases like MBD4 cat , AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. General significance Kinetic analysis of the transfer of DNA between human DNA Glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.
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7 8 dihydro 8 oxoadenine a highly mutagenic adduct is repaired by escherichia coli and human mismatch specific uracil thymine dna Glycosylases
Nucleic Acids Research, 2013Co-Authors: Ibtissam Talhaoui, Christophe Kunz, Primo Schar, Alexander A Ishchenko, Sophie Couve, Murat SaparbaevAbstract:Hydroxyl radicals predominantly react with the C(8) of purines forming 7,8-dihydro-8-oxoguanine (8oxoG) and 7,8-dihydro-8-oxoadenine (8oxoA) adducts, which are highly mutagenic in mammalian cells. The majority of oxidized DNA bases are removed by DNA Glycosylases in the base excision repair pathway. Here, we report for the first time that human thymine-DNA glycosylase (hTDG) and Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) can remove 8oxoA from 8oxoA*T, 8oxoA*G and 8oxoA*C pairs. Comparison of the kinetic parameters of the reaction indicates that full-length hTDG excises 8oxoA, 3,N(4)-ethenocytosine (epsilonC) and T with similar efficiency (k(max) = 0.35, 0.36 and 0.16 min(-1), respectively) and is more proficient as compared with its bacterial homologue MUG. The N-terminal domain of the hTDG protein is essential for 8oxoA-DNA glycosylase activity, but not for epsilonC repair. Interestingly, the TDG status had little or no effect on the proliferation rate of mouse embryonic fibroblasts after exposure to gamma-irradiation. Nevertheless, using whole cell-free extracts from the DNA glycosylase-deficient murine embryonic fibroblasts and E. coli, we demonstrate that the excision of 8oxoA from 8oxoA*T and 8oxoA*G has an absolute requirement for TDG and MUG, respectively. The data establish that MUG and TDG can counteract the genotoxic effects of 8oxoA residues in vivo.
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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.
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excision of hypoxanthine from dna containing dimp residues by the escherichia coli yeast rat and human alkylpurine dna Glycosylases
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Murat Saparbaev, Jacques LavalAbstract:Abstract The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA Glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested.
T Hollis - One of the best experts on this subject based on the ideXlab platform.
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Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase.
Mutation Research-dna Repair, 2000Co-Authors: T Hollis, Tom EllenbergerAbstract:Abstract Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair Glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA–DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a SN1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.
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dna bending and a flip out mechanism for base excision by the helix hairpin helix dna glycosylase escherichia coli alka
The EMBO Journal, 2000Co-Authors: T Hollis, Y. Ichikawa, Tom EllenbergerAbstract:The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an SN1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH Glycosylases can bind to DNA in a similar manner.
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DNA bending and a flip-out mechanism for base excision by the helix- hairpin-helix DNA glycosylase, Escherichia coli AlkA
Embo J, 2000Co-Authors: T Hollis, Y. Ichikawa, Tom EllenbergerAbstract:The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin- helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH Glycosylases can bind to DNA in a similar manner.
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DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA
The EMBO Journal, 2000Co-Authors: T Hollis, Y. Ichikawa, Tom EllenbergerAbstract:The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH Glycosylases can bind to DNA in a similar manner.