Structural Basis for the Inhibition of Human Alkyladenine DNA Glycosylase (AAG) by 3,N4-Ethenocytosine-containing DNA

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N6-ethenoadenine (ϵA) lesions or 3,N4-ethenocytosine (ϵC) lesions, it can only excise ϵA lesions. AAG binds very tightly to DNA containing ϵC lesions, forming an abortive protein-DNA complex; such binding not only shields ϵC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing ϵC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the ϵC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for ϵC versus ϵA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O2 of ϵC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy. Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N6-ethenoadenine (ϵA) lesions or 3,N4-ethenocytosine (ϵC) lesions, it can only excise ϵA lesions. AAG binds very tightly to DNA containing ϵC lesions, forming an abortive protein-DNA complex; such binding not only shields ϵC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing ϵC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the ϵC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for ϵC versus ϵA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O2 of ϵC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy. IntroductionGenotoxic etheno (ϵ)-lesions such as 3,N4-ethenocytosine (ϵC) 5The abbreviations used are: ϵC, 3,N4-ethenocytosine; ϵA, 1,N6-ethenoadenine; AAG, alkyladenine DNA glycosylase; Hx, hypoxanthine; 7-meG, 7-methylguanine; ATL, alkyltransferase-like; AP, apurinic/apyrimidinic; r.m.s.d., root mean square deviation. and 1,N6-ethenoadenine (ϵA) are endogenously generated when DNA is attacked by reactive aldehydes. These reactive compounds are generated as byproducts of lipid peroxidation that is induced by reactive oxygen and nitrogen species. Neutrophils and macrophages generate large quantities of reactive oxygen and nitrogen species in tissues undergoing chronic inflammation (1Coussens L.M. Werb Z. Nature. 2002; 420: 860-867Crossref PubMed Scopus (10953) Google Scholar, 2Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar), and it is widely accepted that such inflammation increases the risk of colon cancer in ulcerative colitis and Crohn disease patients and increases the risk of liver cancer in Wilson disease and hemochromatosis patients (1Coussens L.M. Werb Z. Nature. 2002; 420: 860-867Crossref PubMed Scopus (10953) Google Scholar, 3Nair U. Bartsch H. Nair J. Free Radic. Biol. Med. 2007; 43: 1109-1120Crossref PubMed Scopus (304) Google Scholar). In fact, increased levels of ϵ-lesions in the DNA of tissues undergoing chronic inflammation have been reported for each of these diseases (4Bartsch H. Nair J. Cancer Detect. Prev. 2002; 26: 308-312Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Depending on the type of DNA polymerase, ϵC mispairs with A, T, or C during DNA replication, resulting in both transition and transversion mutations (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar). In contrast, ϵA primarily gives rise to A:T to T:A transversion mutations (6Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). These mutagenic ϵ-lesions are generally removed via the base excision repair pathway, initiated by lesion-specific DNA glycosylases that cleave the N-glycosidic bond between the damaged base and the deoxyribose sugar (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar, 7Zharkov D.O. Cell Mol. Life Sci. 2008; 65: 1544-1565Crossref PubMed Scopus (222) Google Scholar). In humans, several DNA glycosylases can excise ϵC, namely thymine DNA glycosylase, methyl-CpG binding domain protein, and single strand monofunctional uracil DNA glycosylase (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar). In contrast, there is only one DNA glycosylase known to excise ϵA lesions in humans, alkyladenine DNA glycosylase (AAG) (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar, 8Wyatt M.D. Allan J.M. Lau A.Y. Ellenberger T.E. Samson L.D. BioEssays. 1999; 21: 668-676Crossref PubMed Scopus (167) Google Scholar).AAG (also known as MPG and ANPG) has been previously characterized by crystallography. The crystal structures of an N-terminally truncated, but catalytically active, construct of AAG (Δ79AAG), both bound to a pyrrolidine abasic site mimic (Pyr) and bound to an ϵA-containing piece of DNA, suggest a mode by which AAG recognizes a wide range of lesions while still discriminating against undamaged bases (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar). When substrate is bound, AAG can excise the damaged base through acid/base catalysis (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar). A putative catalytic water molecule, revealed by crystallographic studies, is proposed to act as a nucleophile as it is ideally positioned to attack the N-glycosidic bond present between the ϵA base and the deoxyribose sugar (10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar). This water molecule is also in contact with the side chain of Glu-125, which is proposed to be the catalytic base responsible for activating the water for nucleophilic attack. Consistent with this proposal, introduction of an E125Q mutation completely abolishes AAG activity (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar, 11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar). The identity of the general acid is unknown. Interestingly, in this structure of Δ79AAG bound to ϵA:T-containing DNA, the ϵA lesion remained intact (10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar). Later experiments showed that the catalytic activity of Δ79AAG is significantly inhibited in the presence of a variety of divalent metal ions including Mn2+, Zn2+, Ca2+, Cd2+, Ni2+, and most importantly, Mg2+, which was contained in the crystallization buffer (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar, 12Adhikari S. Toretsky J.A. Yuan L. Roy R. J. Biol. Chem. 2006; 281: 29525-29532Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Wang P. Guliaev A.B. Hang B. Toxicol. Lett. 2006; 166: 237-247Crossref PubMed Scopus (37) Google Scholar). However, the structures did not show any Mg2+ ions bound (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar).In addition to repairing ϵA, AAG can repair other reactive oxygen and nitrogen species and alkylation-induced DNA damage, including lesions hypoxanthine (Hx), 1,N2-ethenoguanine, 8-oxoguanine, 3-methyladenine, 7-methylguanine (7-meG), and 3-methylguanine (2Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar, 8Wyatt M.D. Allan J.M. Lau A.Y. Ellenberger T.E. Samson L.D. BioEssays. 1999; 21: 668-676Crossref PubMed Scopus (167) Google Scholar). However, despite this broad substrate specificity, there is a growing list of lesions to which AAG can bind while failing to excise the lesion. In addition to ϵC-containing DNA (14Gros L. Maksimenko A.V. Privezentzev C.V. Laval J. Saparbaev M.K. J. Biol. Chem. 2004; 279: 17723-17730Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), this list now includes 3-methyluracil, 3-ethyluracil, 3-methylthymine, and 3-methylcytosine (15Lee C.Y. Delaney M. J.M. Samson L.D. Biochemistry. PubMed Scopus Google Scholar). This binding can in the formation of abortive between AAG and damaged The abortive complex has been to AAG glycosylase activity in in human and to in (14Gros L. Maksimenko A.V. Privezentzev C.V. Laval J. Saparbaev M.K. J. Biol. Chem. 2004; 279: 17723-17730Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), the of ϵC lesions in In tissues undergoing chronic which have higher ϵC (4Bartsch H. Nair J. Cancer Detect. Prev. 2002; 26: 308-312Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), the formation of abortive significantly the repair of other AAG resulting in the of DNA lesions in addition to ϵC. Interestingly, in ulcerative colitis the colon undergoing chronic inflammation was to have increased AAG an by increased levels of DNA damage M. M. M. Samson L.D. J. 2003; PubMed Scopus Google Scholar). This for the AAG by ϵC understand the structural basis for the inhibition of AAG by ϵC-containing DNA, we have and crystallographic on AAG, a of the that was previously to have catalytic activity (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar, C.Y. Delaney M. J.M. Samson L.D. Biochemistry. PubMed Scopus Google Scholar, P.J. Ellenberger T. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). crystal structure of Δ79AAG bound to a DNA duplex containing in with (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google and that the failure of AAG to activate the leaving group (ϵC) by is the primary for to ϵC from the This structure also that a divalent metal Mn2+, can bind to the base the ϵC sugar and the first structural for the basis for metal inhibition of an in the of through to and excise a wide range of DNA base lesions. was that AAG also has the to and bind a of DNA base lesions that it is of in the ϵC lesion. the binding of AAG to ϵC to the inhibition of catalytic activity in is known to ϵC from and L. for To understand the structural basis for the inhibition of AAG by ϵC-containing DNA, we solved the crystal structure of Δ79AAG bound to a that AAG can bind ϵC-containing DNA, we that the of activity be a of one or of the AAG to ϵC active or it ϵC an binding pocket that the catalytic the binding mode of ϵC in the active site not of the water molecule to act as a nucleophile in the the side chain of the putative catalytic base a that to activate the putative catalytic water AAG be to ϵC, failing to activate it for crystal structure that AAG the ϵC the active site pocket that binds the ϵA the first We also that the putative catalytic water molecule is present in the the as was in the structure of the substrate this water molecule is in contact with Glu-125, as be for The water are ideally positioned to attack the N-glycosidic we that the of AAG to ϵC is to be to a with nucleophilic or the from the that the failure to excise ϵC is to a with leaving group In a and Ellenberger (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google the for the excision of and ϵA lesions by AAG and for excision of the lesion single that the for ϵA and excision a that for the excision of lesions, AAG the of both a general acid and a general base The general base can activate a catalytic water molecule, the general acid is to the of lesions, the lesion base a leaving group (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar). In contrast, the for the excision of only a single to a general that leaving group of is not the base is To the site of the activity of AAG on was with activity on and AAG the of excision the lesion with to is not by AAG, the of the in catalysis for (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar). Although this study was to identify a as the general the crystal structure of a substrate complex a water molecule in contact with the to of Hx, that of ϵA (10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar), the that a water molecule be responsible for the AAG active site is to the of the base through a hydrogen bond between of ϵA and the oxygen of these on the catalytic of of the of and ϵA, it is to the in the ϵC A that ϵA, ϵC has a in the to and be that site as to with to one through it that the failure of AAG to cleave ϵC be to an to activate the ϵC leaving group by AAG is reported to bind and not cleave a of lesions, including 3-methyluracil, 3-ethyluracil, and (15Lee C.Y. Delaney M. J.M. Samson L.D. Biochemistry. PubMed Scopus Google Scholar), this of inhibition be that AAG repair ϵC lesions, it is that AAG binds this lesion The basis for the higher affinity of AAG for the duplex with the substrate can be to an additional hydrogen bond between the side chain of Asn-169 and the O2 of ϵC. of Asn-169 to that this hydrogen bond and completely this binding that this one hydrogen bond is responsible for the higher affinity of ϵC-containing in addition to previously proposed of to between damaged and undamaged Wyatt M.D. Chem. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), Asn-169 to a in the and binding of DNA lesions. is to that Asn-169 is glycosylases (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google inhibition of AAG by divalent metal ions Mn2+, Zn2+, Ca2+, Cd2+, and has been S. Toretsky J.A. Yuan L. Roy R. J. Biol. Chem. 2006; 281: 29525-29532Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Wang P. Guliaev A.B. Hang B. Toxicol. Lett. 2006; 166: 237-247Crossref PubMed Scopus (37) Google Scholar), crystal structure with such an these used in the crystallization (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar). we with the presence of in to the base the ϵC lesion and of to this site to the of the a This is the first that this sugar has been in an AAG In the binding of ϵC to the active site is to that in the the inhibitory of not to be to a large in the active the of the active site be by the presence of the of or DNA that are in catalysis also be by the Although binding of divalent metal to and DNA is it is that we this divalent metal bound to such an site in this protein-DNA We now have a for the of divalent on AAG activity that can be a of the abortive complex in it is to the in forming this complex between and a lesion that it have been S. M. R. B. M. M.D. J.A. Nature. PubMed Scopus Google Scholar). are known to with in DNA repair pathway, nucleotide excision that to present DNA to nucleotide excision repair for repair S. M. R. B. M. M.D. J.A. Nature. PubMed Scopus Google Scholar, S. DNA 2007; PubMed Scopus Google Scholar). the of a single damaged DNA base in the of the it that DNA repair be to lesion bases that repair to an repair Although it is to from show that AAG can with human nucleotide excision repair and M. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). are to abortive with nucleotide excision repair resulting in ϵC but this is the on ATL, not by to excise and lesions M.D. Allan J.M. Lau A.Y. Ellenberger T.E. Samson L.D. BioEssays. 1999; 21: 668-676Crossref PubMed Scopus (167) Google Scholar, L. B. M. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar), the of AAG in DNA repair is complex the of repairing reactive oxygen and nitrogen DNA damage for tissues undergoing chronic a of the of AAG is IntroductionGenotoxic etheno (ϵ)-lesions such as 3,N4-ethenocytosine (ϵC) 5The abbreviations used are: ϵC, 3,N4-ethenocytosine; ϵA, 1,N6-ethenoadenine; AAG, alkyladenine DNA glycosylase; Hx, hypoxanthine; 7-meG, 7-methylguanine; ATL, alkyltransferase-like; AP, apurinic/apyrimidinic; r.m.s.d., root mean square deviation. and 1,N6-ethenoadenine (ϵA) are endogenously generated when DNA is attacked by reactive aldehydes. These reactive compounds are generated as byproducts of lipid peroxidation that is induced by reactive oxygen and nitrogen species. Neutrophils and macrophages generate large quantities of reactive oxygen and nitrogen species in tissues undergoing chronic inflammation (1Coussens L.M. Werb Z. Nature. 2002; 420: 860-867Crossref PubMed Scopus (10953) Google Scholar, 2Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar), and it is widely accepted that such inflammation increases the risk of colon cancer in ulcerative colitis and Crohn disease patients and increases the risk of liver cancer in Wilson disease and hemochromatosis patients (1Coussens L.M. Werb Z. Nature. 2002; 420: 860-867Crossref PubMed Scopus (10953) Google Scholar, 3Nair U. Bartsch H. Nair J. Free Radic. Biol. Med. 2007; 43: 1109-1120Crossref PubMed Scopus (304) Google Scholar). In fact, increased levels of ϵ-lesions in the DNA of tissues undergoing chronic inflammation have been reported for each of these diseases (4Bartsch H. Nair J. Cancer Detect. Prev. 2002; 26: 308-312Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Depending on the type of DNA polymerase, ϵC mispairs with A, T, or C during DNA replication, resulting in both transition and transversion mutations (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar). In contrast, ϵA primarily gives rise to A:T to T:A transversion mutations (6Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). These mutagenic ϵ-lesions are generally removed via the base excision repair pathway, initiated by lesion-specific DNA glycosylases that cleave the N-glycosidic bond between the damaged base and the deoxyribose sugar (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar, 7Zharkov D.O. Cell Mol. Life Sci. 2008; 65: 1544-1565Crossref PubMed Scopus (222) Google Scholar). In humans, several DNA glycosylases can excise ϵC, namely thymine DNA glycosylase, methyl-CpG binding domain protein, and single strand monofunctional uracil DNA glycosylase (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar). In contrast, there is only one DNA glycosylase known to excise ϵA lesions in humans, alkyladenine DNA glycosylase (AAG) (5Gros L. Ishchenko A.A. Saparbaev M. Mutat. Res. 2003; 531: 219-229Crossref PubMed Scopus (77) Google Scholar, 8Wyatt M.D. Allan J.M. Lau A.Y. Ellenberger T.E. Samson L.D. BioEssays. 1999; 21: 668-676Crossref PubMed Scopus (167) Google Scholar).AAG (also known as MPG and ANPG) has been previously characterized by crystallography. The crystal structures of an N-terminally truncated, but catalytically active, construct of AAG (Δ79AAG), both bound to a pyrrolidine abasic site mimic (Pyr) and bound to an ϵA-containing piece of DNA, suggest a mode by which AAG recognizes a wide range of lesions while still discriminating against undamaged bases (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar). When substrate is bound, AAG can excise the damaged base through acid/base catalysis (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar). A putative catalytic water molecule, revealed by crystallographic studies, is proposed to act as a nucleophile as it is ideally positioned to attack the N-glycosidic bond present between the ϵA base and the deoxyribose sugar (10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar). This water molecule is also in contact with the side chain of Glu-125, which is proposed to be the catalytic base responsible for activating the water for nucleophilic attack. Consistent with this proposal, introduction of an E125Q mutation completely abolishes AAG activity (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar, 11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar). The identity of the general acid is unknown. Interestingly, in this structure of Δ79AAG bound to ϵA:T-containing DNA, the ϵA lesion remained intact (10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar). Later experiments showed that the catalytic activity of Δ79AAG is significantly inhibited in the presence of a variety of divalent metal ions including Mn2+, Zn2+, Ca2+, Cd2+, Ni2+, and most importantly, Mg2+, which was contained in the crystallization buffer (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar, 12Adhikari S. Toretsky J.A. Yuan L. Roy R. J. Biol. Chem. 2006; 281: 29525-29532Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Wang P. Guliaev A.B. Hang B. Toxicol. Lett. 2006; 166: 237-247Crossref PubMed Scopus (37) Google Scholar). However, the structures did not show any Mg2+ ions bound (9Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 10Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (214) Google Scholar).In addition to repairing ϵA, AAG can repair other reactive oxygen and nitrogen species and alkylation-induced DNA damage, including lesions hypoxanthine (Hx), 1,N2-ethenoguanine, 8-oxoguanine, 3-methyladenine, 7-methylguanine (7-meG), and 3-methylguanine (2Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1943) Google Scholar, 8Wyatt M.D. Allan J.M. Lau A.Y. Ellenberger T.E. Samson L.D. BioEssays. 1999; 21: 668-676Crossref PubMed Scopus (167) Google Scholar). However, despite this broad substrate specificity, there is a growing list of lesions to which AAG can bind while failing to excise the lesion. In addition to ϵC-containing DNA (14Gros L. Maksimenko A.V. Privezentzev C.V. Laval J. Saparbaev M.K. J. Biol. Chem. 2004; 279: 17723-17730Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), this list now includes 3-methyluracil, 3-ethyluracil, 3-methylthymine, and 3-methylcytosine (15Lee C.Y. Delaney M. J.M. Samson L.D. Biochemistry. PubMed Scopus Google Scholar). This binding can in the formation of abortive between AAG and damaged The abortive complex has been to AAG glycosylase activity in in human and to in (14Gros L. Maksimenko A.V. Privezentzev C.V. Laval J. Saparbaev M.K. J. Biol. Chem. 2004; 279: 17723-17730Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), the of ϵC lesions in In tissues undergoing chronic which have higher ϵC (4Bartsch H. Nair J. Cancer Detect. Prev. 2002; 26: 308-312Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), the formation of abortive significantly the repair of other AAG resulting in the of DNA lesions in addition to ϵC. Interestingly, in ulcerative colitis the colon undergoing chronic inflammation was to have increased AAG an by increased levels of DNA damage M. M. M. Samson L.D. J. 2003; PubMed Scopus Google Scholar). This for the AAG by ϵC understand the structural basis for the inhibition of AAG by ϵC-containing DNA, we have and crystallographic on AAG, a of the that was previously to have catalytic activity (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar, C.Y. Delaney M. J.M. Samson L.D. Biochemistry. PubMed Scopus Google Scholar, P.J. Ellenberger T. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). crystal structure of Δ79AAG bound to a DNA duplex containing in with (11O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google and that the failure of AAG to activate the leaving group (ϵC) by is the primary for to ϵC from the This structure also that a divalent metal Mn2+, can bind to the base the ϵC sugar and the first structural for the basis for metal inhibition of

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.