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• W2042937673 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 between Escherichia 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-Å 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. Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia 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-Å 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. Oxidative DNA damage is generated by a variety of environmental and endogenous agents, including ionizing radiation, certain chemicals, and products of aerobic metabolism (1von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis, London1987Google Scholar). 8-oxoG 1The abbreviations used are: 8-oxoG8-oxo-7,8-dihydroguanine8-oxodG8-oxo-7,8-dihydro-2′-deoxyguanosinedRbldeoxyribitolTth-FpgThermus thermophilus formamidopyrimidine-DNA glycosylaseH2THhelix-two turn-helix motifMe-FaPy2,6-diamino-4-hydroxy-5 N-methylformamidopyrimidineMRmolecular replacement, Nei, endonuclease VIIINthendonuclease IIIOgg18-oxoguanine-DNA glycosylaseAPapurinic/apyrimidinic is one of the most abundant forms of oxidative DNA damage (2Burrows C.J. Muller J.G. Chem. Rev. 1998; 98: 1109-1151Crossref PubMed Scopus (1627) Google Scholar). Due to its ability to form a Hoogstein-type base pair with adenine (3Kouchakdjian M. Bodepudi V. Shibutani S. Eisenberg M. Johnson F. Grollman A.P. Patel D.J. Biochemistry. 1991; 30: 1403-1412Crossref PubMed Scopus (306) Google Scholar), 8-oxoG is miscoding (4Shibutani S. Takeshita M. Grollman A.P. Nature. 1991; 349: 431-434Crossref PubMed Scopus (2047) Google Scholar) and mutagenic, resulting in G→T transversions in bacterial and eukaryotic cells (5Moriya M., Ou, C. Bodepudi V. Johnson F. Takeshita M. Grollman A.P. Mutat. Res. 1991; 254: 281-288Crossref PubMed Scopus (327) Google Scholar, 6Moriya M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1122-1126Crossref PubMed Scopus (433) Google Scholar). The potential harmful effects of this lesion are avoided by base excision repair. In Escherichia coli, formamidopyrimidine-DNA glycosylase (Fpg, EC 3.2.2.23) removes 8-oxoG, Me-FaPy, and several structurally related lesions from damaged DNA (7Tchou J. Kasai H. Shibutani S. Chung M.-H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4690-4694Crossref PubMed Scopus (693) Google Scholar, 8Tchou J. Bodepudi V. Shibutani S. Antoshechkin I. Miller J. Grollman A.P. Johnson F. J. Biol. Chem. 1994; 269: 15318-15324Abstract Full Text PDF PubMed Google Scholar). Fpg is a component of the “GO system” that includes MutY, a mismatch adenine-DNA glycosylase, and MutT, an 8-oxodGTPase (9Michaels M.L. Tchou J. Grollman A.P. Miller J.H. Biochemistry. 1992; 31: 10964-10968Crossref PubMed Scopus (301) Google Scholar,10Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (614) Google Scholar); E. coli strains deficient in any of these genes are strong mutators (11Tajiri T. Maki H. Sekiguchi M. Mutat. Res. 1995; 336: 257-267Crossref PubMed Scopus (318) Google Scholar). 8-oxo-7,8-dihydroguanine 8-oxo-7,8-dihydro-2′-deoxyguanosine deoxyribitol Thermus thermophilus formamidopyrimidine-DNA glycosylase helix-two turn-helix motif 2,6-diamino-4-hydroxy-5 N-methylformamidopyrimidine molecular replacement, Nei, endonuclease VIII endonuclease III 8-oxoguanine-DNA glycosylase apurinic/apyrimidinic Fpg shares significant sequence homology with endonuclease VIII (Nei) of E. coli (12Jiang D. Hatahet Z. Blaisdell J.O. Melamede R.J. Wallace S.S. J. Bacteriol. 1997; 179: 3773-3782Crossref PubMed Google Scholar). Both proteins belong to a family unrelated by sequence or tertiary structure to a larger family of DNA glycosylases, for which the prototype is endonuclease III (Nth) (13Thayer M.M. Ahern H. Xing D. Cunningham R.P. Tainer J.A. EMBO J. 1995; 14: 4108-4120Crossref PubMed Scopus (438) Google Scholar,14Nash H.M. Bruner S.D. Shärer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). The substrate specificity of Fpg differs significantly from Nei (7Tchou J. Kasai H. Shibutani S. Chung M.-H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4690-4694Crossref PubMed Scopus (693) Google Scholar, 8Tchou J. Bodepudi V. Shibutani S. Antoshechkin I. Miller J. Grollman A.P. Johnson F. J. Biol. Chem. 1994; 269: 15318-15324Abstract Full Text PDF PubMed Google Scholar, 15Jiang D. Hatahet Z. Melamede R.J. Kow Y.W. Wallace S.S. J. Biol. Chem. 1997; 272: 32230-32239Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) but closely resembles that of the eukaryotic 8-oxoguanine-DNA glycosylase, Ogg1, a member of the Nth family (14Nash H.M. Bruner S.D. Shärer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 16Rosenquist T.A. Zharkov D.O. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7429-7434Crossref PubMed Scopus (459) Google Scholar,17Zharkov D.O. Rosenquist T.A. Gerchman S.E. Grollman A.P. J. Biol. Chem. 2000; 275: 28607-28617Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Fpg also possesses AP lyase activity, nicking the phosphodiester backbone of DNA at the site of the lesion. Base excision by Fpg is followed immediately by two β-elimination steps, resulting in a single nucleotide gap flanked by phosphate termini (7Tchou J. Kasai H. Shibutani S. Chung M.-H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4690-4694Crossref PubMed Scopus (693) Google Scholar). A Schiff base intermediate, involving Pro-1 of the enzyme and C1′ of the damaged nucleotide, forms early in the reaction sequence and can be reductively trapped by treatment with NaBH4 forming a stable covalent complex (18Tchou J. Grollman A.P. J. Biol. Chem. 1995; 270: 11671-11677Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 19Zharkov D.O. Rieger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-5341Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The mechanism of cleavage is similar to that of Nei (15Jiang D. Hatahet Z. Melamede R.J. Kow Y.W. Wallace S.S. J. Biol. Chem. 1997; 272: 32230-32239Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 20Rieger R.A. McTigue M.M. Kycia J.H. Gerchman S.E. Grollman A.P. Iden C.R. J. Am. Soc. Mass Spectrom. 2000; 11: 505-515Crossref PubMed Scopus (48) Google Scholar), but not to that of Ogg1 where only one β-elimination occurs, and the efficiency of the elimination step is very low compared with base excision (16Rosenquist T.A. Zharkov D.O. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7429-7434Crossref PubMed Scopus (459) Google Scholar, 17Zharkov D.O. Rosenquist T.A. Gerchman S.E. Grollman A.P. J. Biol. Chem. 2000; 275: 28607-28617Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Comparing the structures of Fpg, Nei, and Ogg1 provides a unique opportunity to analyze features of damage recognition and catalysis common to DNA glycosylases/AP lyases. The presence of DNA enhances the analytic power of the model by revealing the precise nature of enzyme-DNA interactions. The structure of the human Ogg1 catalytic domain complexed to DNA has been solved (21Bruner S.D. Norman D.P.G. Verdine G.L. Nature. 2000; 403: 859-866Crossref PubMed Scopus (831) Google Scholar, 22Norman D.P.G. Bruner S.D. Verdine G.L. J. Am. Chem. Soc. 2001; 123: 359-360Crossref PubMed Scopus (76) Google Scholar), as has the structure of E. coli Nei covalently cross-linked to DNA by NaBH4 (23Zharkov D.O. Golan G. Gilboa R. Fernandes A.S. Gerchman S.E. Kycia J.H. Rieger R.A. Grollman A.P. Shoham G. EMBO J. 2002; 23: 789-800Crossref Scopus (154) Google Scholar). The structure of Fpg from Thermus thermophilus HB8 (Tth-Fpg) has recently been solved in the absence of DNA (24Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (138) Google Scholar). Although mechanisms for lesion recognition and catalysis by Fpg have been suggested on the basis of this structure and on earlier biochemical studies of E. coli Fpg (8Tchou J. Bodepudi V. Shibutani S. Antoshechkin I. Miller J. Grollman A.P. Johnson F. J. Biol. Chem. 1994; 269: 15318-15324Abstract Full Text PDF PubMed Google Scholar, 18Tchou J. Grollman A.P. J. Biol. Chem. 1995; 270: 11671-11677Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (138) Google Scholar, 25Rabow L.E. Kow Y.W. Biochemistry. 1997; 36: 5084-5096Crossref PubMed Scopus (76) Google Scholar), many questions remain unanswered regarding the mode of Fpg-DNA interactions and the catalytic reaction mechanism of this important DNA repair protein. To investigate the mechanisms of Fpg-DNA interactions, we have utilized NaBH4 reduction of the Schiff base intermediate to produce a stable covalent cross-link between Fpg and duplex DNA. We crystallized and determined the three-dimensional structure of this complex at 2.1 Å, revealing for the first time the three dimensional precise mode of DNA binding by Fpg as well as several important features of the catalytic mechanism of this enzyme. We used this model to rationalize numerous biochemical observations and site-directed mutagenesis studies of Fpg. The present study provides insight into the structural basis of Fpg-DNA binding, furnishing information essential for a mechanistic understanding of DNA glycosylases. The 13-mers CCAGGA(8-oxoG)GAAGCC and GGCTTCATCCTGG were synthesized by established phosphoramidite chemistry (26Bodepudi V. Shibutani S. Johnson F. Chem. Res. Toxicol. 1992; 5: 608-617Crossref PubMed Scopus (98) Google Scholar) and annealed in a 1:1 ratio. The E. coli fpg gene was amplified from E. coli genomic DNA by PCR using Pfu DNA polymerase (Stratagene); NdeI and BamHI restriction sites were present on the primers. The amplified fragment was inserted into the NdeI-BamHI site of the pET13a plasmid (27Gerchman S.E. Graziano V. Ramakrishnan V. Protein Expr. Purif. 1994; 5: 242-251Crossref PubMed Scopus (64) Google Scholar) and used to transform B834(DE3) E. coli cells. To purify recombinant Fpg, cells from 1 liter of culture, induced with 50 μm isopropyl-1-thio-β-d-galactopyranoside for 6 h at 37 °C, were lysed by treatment with 50 μg/ml lysozyme in Tris/EDTA buffer, pH 8.0, containing 1 mmphenylmethylsulfonyl fluoride. DNA was precipitated with 0.01% polyethyleneimine, 1 m NaCl, and the supernatant was treated with 45% saturated (NH4)2SO4. The pellet was dissolved in Buffer A (20 mm HEPES-NaOH, pH 7.5, 1 mmEDTA, 1 mm dithiothreitol, 200 mm NaCl), loaded on a Fractogel EMD SO3− 650 (Merck) column equilibrated in the same buffer and eluted by a 200–600 mm NaCl gradient in Buffer A. Fractions containing Fpg were diluted, applied to a heparin-Sepharose CL-6B (Amersham Biosciences) column, and eluted with a 200–800% mm NaCl gradient in Buffer A. Fractions of at least 90% purity were used directly for cross-linking. The cross-linking reaction mixture (10 ml), including 100–200 nmol of duplex oligonucleotide, 1–2 mg of Fpg, 25 mm sodium phosphate, pH 6.8, 100 mm NaCl, 1 mm EDTA, and 50 mm NaBH4 was incubated for 30 min at 37 °C and then quenched by adding 400 mm glucose. The sample was loaded onto a Poros HQ 4.6/50 column (PerSeptive Biosystems) equilibrated in 50 mm Tris-HCl, 5 mmMgCl2, 200 mm NaCl, eluted at 460 mm NaCl, concentrated to 1.8–2.0 mg/ml in a Centricon-3 device (Millipore), and used for crystallization experiments. Crystals were obtained by mixing 2-μl volumes of Fpg-DNA complex and reservoir solution (30% (w/v) polyethylene glycol 8000, 0.2 m(NH4)2SO4, 0.1 m sodium cacodylate pH 6.5), and equilibrating the drop with 1 ml of reservoir solution at 15 °C for several days. X-ray diffraction data (Table I) were collected on crystals soaked for ∼3 min in a solution composed of 80% reservoir solution, 20% glycerol and flash-cooled to 100 K under a cold nitrogen gas stream. Data were obtained with a Quantum-4 CCD area detector (ADSC) at the National Synchroton Light Source X26C beamline, Brookhaven National Laboratory, and processed with DENZO and SCALEPACK (28Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar).Table ICrystallographic data collection and refinement parametersData collection Resolution range (Å)34.0–2.1 Space groupP21 a, b, c (Å)80.7, 96.0, 96.3 β96.8 ° Collected reflections (I > 0)310,926 Unique reflections84,396 Completeness (%) (last shell)99.6 (99.7) 〈I/ς(I)〉11.8 Rsym (%) (last shell)6.9 (26.5)Refinement statistics No. atoms: protein8149 DNA2064 water499 Zn4 R-factor (%): work21.4 free1-a5% of the reflections were used as test set for Rfree.26.5 r.m.s.d.: bonds (Å)0.010 angles (°)1.99 Ramachandran plot (%): most favored87.9 disallowed01-a 5% of the reflections were used as test set for Rfree. Open table in a new tab Matthews coefficient calculations suggest four Fpg-DNA monomers in an asymmetric unit. The Fpg-DNA structure was determined by molecular replacement (MR), using REPLACE (29Tong L. Rossmann M.G. Acta Crystallogr. 1990; A46: 783-792Crossref Scopus (256) Google Scholar, 30Tong L. J. Appl. Crystallogr. 1993; 26: 748-751Crossref Scopus (52) Google Scholar) and CNS (31Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. 1998; D54: 905-921Crossref Scopus (16979) Google Scholar). Polar angle self-rotation function calculations resulted in two major peaks, indicating an asymmetric unit of 222 non-crystallographic symmetry. Rotation and translation functions calculations employed all data in the 10.0–4.0-Å resolution range. Structures of Tth-Fpg (24Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (138) Google Scholar) (1EE8) and a recently determined Nei-DNA complex (Ref. 23Zharkov D.O. Golan G. Gilboa R. Fernandes A.S. Gerchman S.E. Kycia J.H. Rieger R.A. Grollman A.P. Shoham G. EMBO J. 2002; 23: 789-800Crossref Scopus (154) Google Scholar, 1von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis, London1987Google ScholarK3W) were combined to form a search model for MR. Calculations imposing non-crystallographic symmetry, implemented in the “locked” cross rotation function in REPLACE, resulted in clear separation between the top and the second peaks, with no overlap between monomers in the unit cell. A translation function search was performed with CNS, using the constructed Fpg-DNA tetramer as a search model and including Patterson correlation refinement (32Brünger A.T. Acta Crystallogr. 1990; A46: 46-57Crossref Scopus (361) Google Scholar) and rigid body refinement for each of the monomers. This search gave a clear MR solution that was used for later stages of refinement. Solvent flipping (33Abrahams J.P. Leslie A.G.W. Acta Crystallogr. 1996; D52: 30-42Crossref Scopus (1142) Google Scholar) and 4-fold averaging, calculated to 2.1 Å resolution, increased the overall figure of merit from 0.42 to 0.89. Electron density maps were calculated from the refined MR phases. An initial model was constructed using the program “O” (34Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. 1991; A47: 110-119Crossref Scopus (13014) Google Scholar), allowing clear determination of most of the structure, including components (e.g. zinc ion and most of the DNA bases) missing from the search model. The model was subjected to simulated annealing and iterative cycles of positional and temperature factor refinement, followed by manual fitting and rebuilding. An overall anisotropic temperature factor and bulk solvent correction factor were applied throughout the calculation. Progress of the refinement was monitored via Rfree (35Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3872) Google Scholar). Strict non-crystallographic symmetry constraints were applied in the initial rounds of refinement. Subsequently, tight non-crystallographic symmetry restraints (force constant of 300 kcal/mol) were applied to allow proper refinement of regions significantly different in structure from that of the search model. All of the DNA and most of the protein residues were identified, except for the loop including residues 217–224 and the side chains of several amino acids remote from the protein-DNA interface. Water molecules were assigned to peaks in the [Fo −Fc] electron density maps larger than 3ς and within potential hydrogen-bonding distance. The figures were prepared using MidasPlus (36Ferrin T.E. Huang C.C. Jarvis L.E. Langridge R. J. Mol. Graphics. 1988; 6 (27, 36–37): 13Crossref Scopus (929) Google Scholar), MOLSCRIPT (37Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), BOBSCRIPT (38Esnouf R.M. J. Mol. Graphics. 1997; 15: 132-134Crossref Scopus (1795) Google Scholar), Raster3D (39Merrit E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3878) Google Scholar), and GRASP (40Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar). 3DNA (41Lu X.-J. Shakked Z. Olson W.K. J. Mol. Biol. 2000; 300: 819-840Crossref PubMed Scopus (307) Google Scholar) was used to calculate various DNA structural parameters. A model of Fpg complexed with the DNA duplex containing an everted 8-oxodG residue was built based on the current structure and the structure of 8-oxoG-containing DNA from the Ogg1-DNA complex (1EBM, Ref. 21Bruner S.D. Norman D.P.G. Verdine G.L. Nature. 2000; 403: 859-866Crossref PubMed Scopus (831) Google Scholar). The missing residues 217–224 were modeled based on the corresponding loop of Tth-Fpg (1EE8, Ref. 24Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (138) Google Scholar). The model was subjected to a series of energy minimization steps using the Discover module of Insight II (Accelrys) until the root mean-squared gradient was smaller than 0.001 kcal/(mol·Å). All energy optimizations were performed using the AMBER force field (42Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta S., Jr. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4895) Google Scholar) with a distance-dependent dielectric constant of 4r, using the steepest descent and conjugate gradient methods. The phosphorus atoms of the DNA and the Cα atoms of the protein, except for the newly built loop, were restrained with harmonic forces. To find a conformation with lower energy for the missing loop and the everted 8-oxodG, we also performed simulated annealing molecular dynamic runs from 1000 to 298 K. The conservation of residues within the Nei and Fpg subfamilies of the Fpg family was analyzed using the AMAS algorithm (43Livingstone C.D. Barton G.J. Comput. Appl. Biosci. 1993; 9: 745-756PubMed Google Scholar) and the cluster of orthologous groups data base (44Tatusov R.L. Natale D.A. Garkavtsev I.V. Tatusova T.A. Shankavaram U.T. Rao B.S. Kiryutin B. Galperin M.Y. Fedorova N.D. Koonin E.V. Nucleic Acids Res. 2001; 29: 22-28Crossref PubMed Scopus (1560) Google Scholar), as described elsewhere (23Zharkov D.O. Golan G. Gilboa R. Fernandes A.S. Gerchman S.E. Kycia J.H. Rieger R.A. Grollman A.P. Shoham G. EMBO J. 2002; 23: 789-800Crossref Scopus (154) Google Scholar). The overall structure of Fpg comprises two domains connected by a hinge polypeptide (Fig. 1, a and b) and resembles that of Tth-Fpg (24Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (138) Google Scholar) and E. coli Nei (23Zharkov D.O. Golan G. Gilboa R. Fernandes A.S. Gerchman S.E. Kycia J.H. Rieger R.A. Grollman A.P. Shoham G. EMBO J. 2002; 23: 789-800Crossref Scopus (154) Google Scholar). The N-terminal domain contains eight β-strands, forming a β-sandwich with two α-helices parallel to its edges. The C-terminal domain includes four α-helices, two of which, αD and αE, form the helix-two turn-helix (H2TH) motif, and two β-strands that form a β-hairpin zinc finger. The excised base, 8-oxoG, is not retained in the crystal. The ring-opened deoxyribitol moiety (dRbl), formed after base excision and NaBH4 reduction, is everted from the helix with C1′ bound covalently to Nα of Pro-1 (Fig. 2), as suggested by biochemical experiments identifying Pro-1 as the residue involved in Schiff base formation (18Tchou J. Grollman A.P. J. Biol. Chem. 1995; 270: 11671-11677Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 19Zharkov D.O. Rieger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-5341Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). DNA is severely kinked at the lesion point (roll angle, 66°), and the minor groove is significantly widened. Except at the lesion site, the DNA is essentially B-form. As commonly observed in DNA-binding proteins, the DNA-binding groove is positively charged (Fig. 3).Figure 3The solvent-accessible surface of Fpg, colored according to electrostatic potential and demonstrating the positive DNA binding cleft. The bound 13-mer DNA duplex is superimposed on the positively charged surface as a stick model (yellow). The negatively charged binding pocket is clearly seen in the center of the groove, containing the modeled 8-oxoG (inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Binding to DNA involves extensive interactions between Fpg and DNA (Fig. 4). A hydrogen bond network involving all loops that face DNA comprises 2512 Å2 of contact surface area. This relatively large buried surface area is consistent with the heat capacity change reported for Fpg binding to a lesion-containing duplex. 2C. A. Minetti, D. P. Remeter, E. G. Plum, and K. J. Breslauer, unpublished observations. A similar value for binding-induced burial of previously exposed solvent accessible surface area (2268 Å2) has been reported for Ogg1 (21Bruner S.D. Norman D.P.G. Verdine G.L. Nature. 2000; 403: 859-866Crossref PubMed Scopus (831) Google Scholar); significantly higher than for human alkylpurine-DNA glycosylase (1034 Å2) (45Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) and uracil-DNA glycosylase (700 Å2) (46Parikh S.S. Mol C.D. Slupphaug G. Bharati S. Krokan H.E. Tainer J.A. EMBO J. 1998; 17: 5214-5226Crossref PubMed Scopus (421) Google Scholar). Fpg mainly contacts the damaged strand 3′ to dRbl (P0, P−1, P−2) via side chains and backbone hydrogen atoms of the highly conserved Lys-56, His-70, Asn-168, Tyr-236, and Arg-258 residues. Fpg binds DNA in the minor groove; the damaged base is extruded from the helix through the major groove (Fig. 3). The complementary strand is held in position largely through Watson-Crick bonds; interactions with the enzyme are few, and except for His-89, the amino acids involved are not conserved. Asn-168, Arg-258, and Lys-56 contact P0, P−1, and P−2, stabilizing the complex (Fig. 5 a). Asn-168 is part of the H2TH motif and forms bonds through backbone and side chain amides to P−1 and P0, respectively. The zinc finger forms four hydrogen bonds with the phosphodiester backbone, three of which involve Arg-258 (two to P0, one to P−1) and one via Gln-257, to P(3). Lys-56, located on the β2-β3 loop, forms hydrogen bonds with P−1 and P−2. Extrusion of the damaged base, which facilitates its binding in the enzyme active site, is a common structural feature of DNA glycosylases (21Bruner S.D. Norman D.P.G. Verdine G.L. Nature. 2000; 403: 859-866Crossref PubMed Scopus (831) Google Scholar, 45Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 47Slupphaug G. Mol C.D. Kavli B. Arvai A.S. Krokan H.E. Tainer J.A. Nature. 1996; 384: 87-92Crossref PubMed Scopus (485) Google Scholar, 48Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (206) Google Scholar). Eversion of deoxyribose is achieved by rotation around the P-O5′ and O3′-P bonds. The geometry of Fpg-induced DNA kinking differs from that observed for uracil-DNA glycosylase (46Parikh S.S. Mol C.D. Slupphaug G. Bharati S. Krokan H.E. Tainer J.A. EMBO J. 1998; 17: 5214-5226Crossref PubMed Scopus (421) Google Scholar, 47Slupphaug G. Mol C.D. Kavli B. Arvai A.S. Krokan H.E. Tainer J.A. Nature. 1996; 384: 87-92Crossref PubMed Scopus (485) Google Scholar) and Ogg1 (21Bruner S.D. Norman D.P.G. Verdine G.L. Nature. 2000; 403: 859-866Crossref PubMed Scopus (831) Google Scholar), which “pinch” the DNA backbone at the extrusion site, decreasing the normal distance (∼12 Å in B-DNA) between P−1 and P1 by 30–50%. Similar to E. coli and human alkylpurine-DNA glycosylases complexed with DNA (45Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 48Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (206) Google Scholar), Fpg does not compress the phosphodiester backbone (P−1–P1is 11.4 Å). Thus, base eversion is not absolutely dependent on strain induced by massive backbone compression (46Parikh S.S. Mol C.D. Slupphaug G. Bharati S. Krokan H.E. Tainer J.A. EMBO J. 1998; 17: 5214-5226Crossref PubMed Scopus (421) Google Scholar, 47Slupphaug G. Mol C.D. Kavli B. Arvai A.S. Krokan H.E. Tainer J.A. Nature. 1996; 384: 87-92Crossref PubMed Scopus (485) Google Scholar). Nucleotide eversion effected by Fpg may be achieved by “holding” the flanking phosphates with Lys-56, Asn-168, and Arg-258, coupled with intercalation of hydrophobic residues into duplex DNA (49Werner R.M. Jiang Y.L. Gordley R.G. Jagadeesh G.J. Ladner J.E. Xiao G. Tordova M. Gilliland G.L. Stivers J.T. Biochemistry. 2000; 39: 12585-12594Crossref PubMed Scopus (59) Google Scholar) (Fig. 5,a and b). The energy required for extruding a single base from kinked double helical DNA has been estimated at ∼3 kcal/mol (50van Aalten D.M.F. Erlanson D.A. Verdine G.L. Joshua-Tor L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11809-11814Crossref PubMed Scopus (32) Google Scholar), a barrier readily attained through noncovalent enzyme-DNA interactions. Eversion of dRb1, coupled with loss of a base, creates a substantial gap between opposite strands, reflected in the 14.7 Å distance between C1′ of dRb1 and C(0), as compared with 10.5 Å in B-DNA. The gap is filled by the hydrophobic residues Met-73, Arg-108, and Phe-110 (Fig. 5 b). Met-73, part of the β4-β5 loop, enters the helix through the minor groove, occupying the position vacated by the extruded base. Arg-108 and Phe-110 are located on the β7-β8 loop. Phe-110, wedged between C(1)and C(0), engages in face-to-face π interactions (51Burley S.K. Petsko G.A. Science. 1985; 229: 23-28Crossref PubMed Scopus (2256) Google Scholar) with the C(1) pyrimidine ring. Unstacking of these bases may contribute significantly to DNA kinking. C(0) remains intrahelical, stabilized by hydrogen bonds from O2 and N3 to Arg-108, contributing to Fpg specificity through opposite base recognition (Fig. 5 b). The position of dRb1 is fixed by the Nα–C1′ covalent bond and the absolutely conserved Glu-2, which forms a hydrogen bond with O4′. Glu-2 is stabilized by hydrogen bonds to backbone amides of Ile-169 and Gly-167 located in the H2TH αE helix. Because the E2Q mutation inactivates the glycosylase activity of Fpg, but does not affect its AP l" @default.
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• W2042937673 date "2002-05-01" @default.
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• W2042937673 title "Structure of Formamidopyrimidine-DNA Glycosylase Covalently Complexed to DNA" @default.
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