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• W2020678474 abstract "The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X2-His-X16-Cys-X2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity. The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X2-His-X16-Cys-X2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity. Oxidative DNA damage has been implicated in mutagenesis and is suggested to be involved in the etiology of aging and many diseases including cancer (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5370) Google Scholar, 2Breen A.P. Murphy J.A. Free Radic. Biol. Med. 1995; 18: 1033-1077Crossref PubMed Scopus (914) Google Scholar). Repair of oxidatively damaged bases in all of the organisms occurs primarily via the DNA base excision repair pathway, which is initiated with the excision of damaged bases by DNA glycosylases (3Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-16Crossref PubMed Scopus (722) Google Scholar). Until recently, only two DNA glycosylases, NTH1 (endonuclease III homolog) and OGG1 (8-oxoguanine DNA glycosylase), have been characterized in mammals, which are responsible for repair of oxidized pyrimidine and purine base lesions, respectively. Both OGG1 and NTH1, orthologs of the Escherichia coli glycosylase Nth, utilize an internal Lys residue as the active site nucleophile and carry out β-elimination at the abasic (AP) 1The abbreviations used are: AP, abasic; DTT, dithiothreitol; Fpg, FapyG-DNA glycosylase; Nei, endonuclease VIII; NEIL, Nei-like; PBS, phosphate-buffered saline; WT, wild type; B11, 11-nt bubble; PDB, Protein Data Bank; 5-OHU·G, 5-hydroxyuracil.1The abbreviations used are: AP, abasic; DTT, dithiothreitol; Fpg, FapyG-DNA glycosylase; Nei, endonuclease VIII; NEIL, Nei-like; PBS, phosphate-buffered saline; WT, wild type; B11, 11-nt bubble; PDB, Protein Data Bank; 5-OHU·G, 5-hydroxyuracil. site generated after base removal (4Ikeda S. Biswas T. Roy R. Izumi T. Boldogh I. Kurosky A. Sarker A.H. Seki S. Mitra S. J. Biol. Chem. 1998; 273: 21585-21593Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 5Lu R. Nash H.M. Verdine G.L. Curr. Biol. 1997; 7: 397-407Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). However, E. coli has two other oxidized base-specific DNA glycosylases, namely MutM/Fpg and its paralog, Nei (6Boiteux S. J. Photochem. Photobiol. B Biol. 1993; 19: 87-96Crossref PubMed Scopus (133) Google Scholar, 7Jiang D. Hatahet Z. Blaisdell J.O. Melamede R.J. Wallace S.S. J. Bacteriol. 1997; 179: 3773-3782Crossref PubMed Google Scholar), which utilize the N-terminal Pro as the active site nucleophile (8Zharkov 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 (175) Google Scholar) and carry out βδ-elimination at the AP site after excising the base lesion. We and others (9Hazra T.K. Izumi T. Boldogh I. Imhoff B. Kow Y.W. Jaruga P. Dizdaroglu M. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3523-3528Crossref PubMed Scopus (434) Google Scholar, 10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 11Bandaru V. Sunkara S. Wallace S.S. Bond J.P. DNA Repair (Amst.). 2002; 1: 517-529Crossref PubMed Scopus (282) Google Scholar, 12Rosenquist T.A. Zaika E. Fernandes A.S. Zharkov D.O. Miller H. Grollman A.P. DNA Repair (Amst.). 2003; 2: 581-591Crossref PubMed Scopus (162) Google Scholar, 13Takao M. Kanno S. Kobayashi K. Zhang Q.M. Yonei S. van der Horst G.T. Yasui A. J. Biol. Chem. 2002; 277: 42205-42213Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) recently discovered and characterized two other mammalian DNA glycosylases and named these NEIL1 and NEIL2 (Nei-like-1 and -2), which are orthologs of E. coli Fpg/Nei (9Hazra T.K. Izumi T. Boldogh I. Imhoff B. Kow Y.W. Jaruga P. Dizdaroglu M. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3523-3528Crossref PubMed Scopus (434) Google Scholar, 10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 11Bandaru V. Sunkara S. Wallace S.S. Bond J.P. DNA Repair (Amst.). 2002; 1: 517-529Crossref PubMed Scopus (282) Google Scholar, 12Rosenquist T.A. Zaika E. Fernandes A.S. Zharkov D.O. Miller H. Grollman A.P. DNA Repair (Amst.). 2003; 2: 581-591Crossref PubMed Scopus (162) Google Scholar, 13Takao M. Kanno S. Kobayashi K. Zhang Q.M. Yonei S. van der Horst G.T. Yasui A. J. Biol. Chem. 2002; 277: 42205-42213Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Both NEILs use the N-terminal Pro as the active site and function as a DNA glycosylase/AP lyase to carry out βδ-elimination (9Hazra T.K. Izumi T. Boldogh I. Imhoff B. Kow Y.W. Jaruga P. Dizdaroglu M. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3523-3528Crossref PubMed Scopus (434) Google Scholar, 10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The recombinant NEILs are active in excising a variety of oxidatively damaged bases but show significant differences in substrate preference. NEIL1 prefers reactive oxygen species-derived pyrimidines lesions and also efficiently removes FapyG and FapyA, the ring-opened oxidation products of purines (9Hazra T.K. Izumi T. Boldogh I. Imhoff B. Kow Y.W. Jaruga P. Dizdaroglu M. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3523-3528Crossref PubMed Scopus (434) Google Scholar, 12Rosenquist T.A. Zaika E. Fernandes A.S. Zharkov D.O. Miller H. Grollman A.P. DNA Repair (Amst.). 2003; 2: 581-591Crossref PubMed Scopus (162) Google Scholar). NEIL2 removes oxidized pyrimidine substrates from duplex DNA but is more efficient in excising oxidized bases when they are located in a DNA bubble structure (14Dou H. Mitra S. Hazra T.K. J. Biol. Chem. 2003; 278: 49679-49684Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar).NEIL2 shares overall identity of 32 and 27% with Fpg and Nei, respectively, and the key residues of the E. coli enzymes, particularly the N-terminal PE(L/G)P(E/L) motif, are completely conserved in NEIL2 (10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). In contrast to the Nth family, the Fpg/Nei family utilizes two DNA binding motifs, a helix-two-turn-helix (15Thayer M.M. Ahern H. Xing D. Cunningham R.P. Tainer J.A. EMBO J. 1995; 14: 4108-4120Crossref PubMed Scopus (431) Google Scholar) and a zinc finger motif (16Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (136) Google Scholar). Fpg and Nei share significant homology with each other including the sequence of the zinc finger motif, which is of the C4 type (17Zharkov D.O. Shoham G. Grollman A.P. DNA Repair (Amst.). 2003; 2: 839-862Crossref PubMed Scopus (125) Google Scholar). The zinc finger motifs are often involved in specific DNA recognition and have been identified in many DNA-binding proteins, transcription factors, and products of developmental control genes (18Brown R.S. Sander C. Argos P. FEBS Lett. 1985; 186: 271-274Crossref PubMed Scopus (308) Google Scholar, 19Miller J. McLachlan A.D. Klug A. EMBO J. 1985; 4: 1609-1614Crossref PubMed Scopus (1664) Google Scholar, 20Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1246) Google Scholar, 21Falchuk K.H. Prog. Clin. Biol. Res. 1993; 380: 91-111PubMed Google Scholar). Furthermore, several proteins associated with DNA repair, such as Xeroderma Pigmentosum complementation group A, poly(ADP-ribose) polymerase, and replication-associated protein A (RPA), have been shown to contain zinc finger domains (22Tanaka K. Miura N. Satokata I. Miyamoto I. Yoshida M.C. Satoh Y. Kondo S. Yasui A. Okayama H. Okada Y. Nature. 1990; 348: 73-76Crossref PubMed Scopus (339) Google Scholar, 23Menissier-de Murcia J. Molinete M. Gradwohl G. Simonin F. de Murcia G. J. Mol. Biol. 1989; 210: 229-233Crossref PubMed Scopus (157) Google Scholar, 24Mazen A. Menissier-de Murcia J. Molinete M. Simonin F. Gradwohl G. Poirier G. de Murcia G. Nucleic Acids Res. 1989; 17: 4689-4698Crossref PubMed Scopus (82) Google Scholar, 25Bochkareva E. Korolev S. Bochkarev A. J. Biol. Chem. 2000; 275: 27332-27338Abstract Full Text Full Text PDF PubMed Google Scholar). In E. coli, the UvrA protein, which is involved in DNA damage recognition during nucleotide excision repair, also possesses zinc finger domain (26Navaratnam S. Myles G.M. Strange R.W. Sancar A. J. Biol. Chem. 1989; 264: 16067-16071Abstract Full Text PDF PubMed Google Scholar, 27Doolittle R.F. Johnson M.S. Husain I. Van Houten B. Thomas D.C. Sancar A. Nature. 1986; 323: 451-453Crossref PubMed Scopus (111) Google Scholar). Identification of zinc finger motifs in the ever-growing number of DNA-binding proteins is based primarily on the presence of conserved Cys or His residues and the spacing between them, which may be critical in recognition of specific double-stranded DNA sequences.Here we show that NEIL2 possesses a single unusual CHCC-type zinc finger motif at its C terminus, which is distinct from that of Nei/Fpg, and that this motif is essential for maintaining the structural integrity and activity of NEIL2.EXPERIMENTAL PROCEDURESExpression of Wild-type (WT) and Mutant NEIL2 Polypeptides—The WT full-length NEIL2 was cloned between the NdeI/XhoI sites of the expression plasmid pRSETB (Invitrogen) (10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The NEIL2 mutants (C291S, H295A, C315S, C318S, and R310A) were generated using a site-directed mutagenesis kit (Stratagene), and their authenticity was confirmed by direct DNA sequencing.Log-phase cultures of E. coli DE884 mutM nei were transformed with expression plasmids of WT and mutant NEIL2 and then induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside at 16 °C for 16 h. After centrifugation, the cell pellets were suspended in a lysis buffer containing 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 100 μg/ml lysozyme, 5 mm dithiothreitol (DTT), and protease inhibitor mixture. After sonication and centrifugation, the supernatant was used for Western blot analysis or activity assay by trapping analysis (10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar).Purification of Anti-NEIL2 Antibody and Western Blot Analysis— Polyclonal anti-NEIL2 antibodies were purified from rabbit antisera produced by Alpha Diagnostics (San Antonio, TX) by affinity chromatography on Sepharose 4B (Amersham Biosciences) covalently coupled to NEIL2. NEIL2-specific IgG was eluted with glycine-HCl, pH 2.8, and immediately neutralized with 0.1 volume of 1 m Tris base and stored at -80 °C after concentration (Amicon) and dialysis in phosphate-buffered saline (PBS). Lysates of E. coli (2 μg) expressing WT and mutants of NEIL2 were used for immunoblot analysis using ECL system as per manufacturer's protocol (Amersham Biosciences).Analysis of Trapped NEIL2 Complexes—A 32P-labeled duplex oligomer (100 fmol) containing 5-OHU·G was incubated with lysates (1 μg) of E. coli expressing WT or mutant NEIL2 in 15 μl of assay buffer in the presence of 25 mm NaCNBH3 at 37 °C for 30 min. The trapped complexes were then separated by SDS-PAGE (12% polyacrylamide) as described previously (10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar).Purification of Wild-type and Mutant NEIL2—The recombinant WT NEIL2 polypeptide was purified as before (10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Two of the mutant proteins, namely C291S and H295A, were also purified similarly with some protocol modifications. After Polymin P precipitation, the ammonium sulfate-fractionated pellets were dialyzed in buffer A (25 mm Tris-HCl, pH 7.5, 10% glycerol, and 0.5 mm DTT) containing 100 mm NaCl and passed through 5-ml Q- and SP-Sepharose (Amersham Biosciences) columns attached in tandem, which were then washed with 120 mm NaCl. The mutant NEIL2 proteins in the flow-through were concentrated in an Amicon filter and loaded onto a 25-ml Superdex 75 column. The fractions eluted from Superdex were further purified by fast protein liquid chromatography on Mono Q using NaCl gradient (20–200 mm) in buffer A. The NEIL2 mutants eluted at around 75 mm NaCl. The other two mutant proteins, C291S and C315S, were subcloned into a C-terminal His tag-containing vector, pET22b (Novagen). After induction and sonication as before, the cells were spun down at 13 K and the supernatant was loaded onto a Ni2+-nitrilotrioacetate-agarose column (Qiagen) previously equilibrated with buffer B (40 mm Tris-HCl, pH 7.5, and 1 m NaCl). After washing with buffer B, the His-tagged mutant proteins were eluted with an imidazole gradient (20–200 mm imidazole in buffer B). After elution, the peak enzyme fractions were dialyzed against buffer C (25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm DTT, and 10% glycerol). The enzymes were further purified by fast protein liquid chromatography on a Mono Q column like the untagged mutants. The arginine mutant R310Q was also His-tagged in the C terminus and purified from the extract of plasmid-bearing E. coli by affinity chromatography on Ni2+-nitrilotrioacetate-agarose (Qiagen). After elution with 150 mm imidazole, the enzyme was dialyzed against 25 mm Tris-HCl, pH 7.5, and 50 mm NaCl and finally purified on a Mono S column. Purified preparations of WT or mutant NEIL2 proteins were never frozen and were stored at -20 °C in PBS containing 50% glycerol.Incision Assay with 5-OHU-containing Bubble Oligomer—DNA strand cleavage at the abasic (AP) site after damaged base excision by NEIL2 occurs because of its intrinsic AP lyase activity. We have shown previously that NEIL2 has higher activity when the lesion is inside a bubble in an otherwise duplex oligomer (14Dou H. Mitra S. Hazra T.K. J. Biol. Chem. 2003; 278: 49679-49684Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). The strand incision by NEIL2 was used for its assay using an oligomer containing 5-OHU in the middle of unpaired 11-nt bubble (B11) as described previously (14Dou H. Mitra S. Hazra T.K. J. Biol. Chem. 2003; 278: 49679-49684Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). The 51-mer oligomer, 32P-labeled at the 5′ terminus of the lesion-containing strand, was incubated with NEIL2 (WT and mutants) at 37 °C for 15 min in a 15-μl reaction mixture containing 40 mm Hepes, pH 7.5, 50 mm KCl, 100 μg/ml bovine serum albumin, and 5% glycerol. After the reaction was stopped with 80% formamide and 20 mm NaOH, the cleaved oligomers were separated by denaturing gel electrophoresis in 15% polyacrylamide containing 7 m urea in 90 mm Tris borate, pH 8.3, and 2 mm EDTA. The radioactivity in the substrate and cleaved product was analyzed by PhosphorImager (Amersham Biosciences).Electrophoretic Gel Mobility Shift Assay—The wild-type and mutant NEIL2 (C291S, H295A, C315S, C318S, and R310Q) were incubated with a 5′-32P-labeled 5-OHU-containing bubble oligomer (5-OHU·B11) in 10 μl of buffer containing 25 mm Hepes-KOH, pH 7.6, 50 mm NaCl, 0.5 mm EDTA, 0.5 mm DTT, 10 μg/ml poly(dI-dC), and 12% glycerol at 20 °C for 15 min followed by electrophoresis in 6% nondenaturing polyacrylamide gel containing 25 mm Tris-HCl, pH 7.5, 55 mm borate, and 0.6 mm EDTA, pH 7.4, at room temperature. The radioactivity in the shifted DNA protein complex was analyzed by PhosphorImager.Quantitation of Zinc in NEIL2—The zinc content of wild-type and mutant NEIL2 polypeptides was determined by inductively coupled plasma mass spectrometry. The enzymes were dialyzed against PBS prior to analysis of Zn2+ using a calibration curve with known amounts of Zn2+. The Zn content of the enzymes was corrected for contamination in the dialysis buffer.Circular Dichroism Spectroscopy—All of the CD spectra were collected from proteins after dialysis and filtration in an AVIV 60DS spectrometer at 25 °C. An average of three scans of the spectra (250–200 nm) was used to obtain the final data. The molar ellipticity (θ) was calculated using Equation 1,θ=θobs×10−3×MWC×l×n×10−2deg dmol−1cm2(Eq. 1) where θobs is the observed ellipticity, MW is molecular weight, C is concentration (mg/ml), l is the path length of the cuvette in centimeters, and n refers to the number of residues. Protein concentrations were determined by the Bradford assay using bovine serum albumin as the standard.Molecular Modeling of 192–319 Residues of NEIL2—The sequence of NEIL2 with the potential DNA binding region (residues 192–319) was used as the seed sequence to search for a suitable template using BIOSERVER (meta server located at bioserv.cbs.cnrs.fr/). BIOSERVER submits to fold-recognition servers like 3D-PSSM (28Bates P.A. Kelley L.A. MacCallum R.M. Sternberg M.J. Proteins. 2001; 5: 39-46Crossref PubMed Scopus (489) Google Scholar), mGenThreader (29McGuffin L.J. Bryson K. Jones D.T. Bioinformatics. 2000; 16: 404-405Crossref PubMed Scopus (2665) Google Scholar), Sam-T99 (30Karplus K. Barrett C. Hughey R. Bioinformatics. 1998; 14: 846-856Crossref PubMed Scopus (905) Google Scholar), and (Protein Data Bank (PDB)-BLAST. The results from different servers are parsed automatically, and the TITO program is used to evaluate most compatible template (31Labesse G. Mornon J. Bioinformatics. 1998; 14: 206-211Crossref PubMed Scopus (63) Google Scholar). The crystal structure of E. coli formamidopyrimidine-DNA glycosylase (Fpg/MutM, PDB code 1k82) (32Gilboa R. Zharkov D.O. Golan G. Fernandes A.S. Gerchman S.E. Matz E. Kycia J.H. Grollman A.P. Shoham G. J. Biol. Chem. 2002; 277: 19811-19816Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) was selected as the most favorable template with a TITO score of -68,248 (PDB-BLAST score for this template: 1e-37; SAM-T99 score: 4.37e-42; 3DPSSM score: 2.7e-3; and mGenThreader score for 1ee8 template: 3e-4). The initial alignment was improved to minimize gaps in the β or helix regions, and the final alignment had an identity of 30% to the template. Distance and dihedral constraints were extracted from the template using the geometry extraction program EXDIS, available in the homology modeling package MPACK (33Mathura V.S. Soman K.V. Varma T.K. Braun W. J. Mol. Model. 2003; 9: 298-303Crossref PubMed Scopus (11) Google Scholar, 34Ivanciuc O. Oezguen N. Mathura V.S. Schein C.H. Xu Y. Braun W. Curr. Med. Chem. 2004; 11: 583-593Crossref PubMed Scopus (39) Google Scholar, 35Hasan R.J. Pawelczyk E. Urvil P.T. Venkatarajan M.S. Goluszko P. Kur J. Selvarangan R. Nowicki S. Braun W.A. Nowicki B.J. Infect. Immun. 2002; 70: 4485-4493Crossref PubMed Scopus (35) Google Scholar). Structurally conserved regions or the fragments defined by excluding gaps in the pairwise alignment were used to extract geometric constraints. Upper and lower distance constraints were defined either by adding or subtracting a threshold of 0.25 Å to the actual distance for matching side-chain and main-chain (backbone) atoms. To position the side chains of Cys-291, His-295, Cys-315, and Cys-318, additional distance constraints among heavy side chain atoms were extracted from a CHCC-type zinc finger found in the DNA-binding domain of RAG1 (PDB code 1rmd, residues Cys-41, His-43, Cys-61, Cys-64). Upper- and lower-bound dihedral angle constraints were defined by adding or subtracting 5°. A total of 30 distance constraints per atom were extracted from the matching regions of the template. Models were generated using the distance geometry program DIAMOD. A few cycles of constrained energy minimization were applied using the program FANTOM, which minimizes constraint energies by successive application of quasi-Newton and Newton-Raphson minimizers (36Schaumann T. Braun W. Wuthrich K. Biopolymers. 1990; 29: 679-694Crossref Scopus (96) Google Scholar), using the ECEPP/2 forcefield. The conformational energy of the model after energy minimization was -410 kcal/mol.RESULTSExpression and Activity of WT and Mutant NEIL2 in Crude E. coli Extracts—Sequence alignment of NEIL2 with Fpg and Nei predicted that NEIL2 is a zinc finger protein with a CHCC-type motif near the C terminus. Cys-291, His-295, Cys-315, and Cys-318 are candidate residues for coordinating Zn2+ (Fig. 1). This motif is distinct from the zinc finger motifs of Nei/Fpg, which are of the CCCC type (17Zharkov D.O. Shoham G. Grollman A.P. DNA Repair (Amst.). 2003; 2: 839-862Crossref PubMed Scopus (125) Google Scholar). Single point mutants of NEIL2, C291S, H295A, C315S, and C318S were expressed in E. coli, and their expression was monitored by Western analysis (Fig. 2A). The mutations did not affect the expression of NEIL2 in E. coli as indicated by the presence of a protein band of the predicted size in each induced bacterial lysate. In E. coli expressing the H295A mutant, a protein with slight slower migration was observed, which was not present (lane 2) in the control extract of E. coli, expressing the empty vector. Thus this band should be the H295A mutant of NEIL2. This analysis also underscored the strong specificity of the antibody.Fig. 2Expression and activity of WT and mutant NEIL2 in crude E. coli extracts. A, Western analysis of soluble extract of E. coli expressing wild-type and mutant NEIL2. An arrow indicates the position of migration of the purified recombinant NEIL2 protein (lane 1). B, analysis of trapped complexes with extracts of E. coli expressing WT and mutant NEIL2. 5′-32P-labeled 5-OHU·G (0.1 pmol) was incubated with no protein, recombinant NEIL2, vector control, and E. coli extracts in the presence of NaCNBH3 before SDS-PAGE. The positions of free DNA and trapped complexes of purified NEIL2 and E. coli Nth (as marker) are indicated. Other details are given under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT)All of the DNA glycosylases/AP lyases, regardless of their substrate preference, form transient Schiff bases with free AP site in DNA, which could be reduced with NaCNBH3 (or NaBH4) to form a stable “trapped complex” (37McCullough A.K. Dodson M.L. Lloyd R.S. Annu. Rev. Biochem. 1999; 68: 255-285Crossref PubMed Scopus (328) Google Scholar, 38Hill J.W. Hazra T.K. Izumi T. Mitra S. Nucleic Acids Res. 2001; 29: 430-438Crossref PubMed Scopus (350) Google Scholar). We have shown earlier that NEIL2, similar to other MutM/Nei type enzymes, is inactivated when the N-terminal Pro, the active site nucleophile, is blocked or eliminated (10Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Fig. 2B shows SDS-PAGE separation of 32P-labeled trapped complexes generated with extracts of mutM nei E. coli harboring WT or mutant NEIL2 expression plasmids after incubation of a 5-OHU·G-containing oligomer. Because the mobility of such complexes reflects the size of the DNA glycosylase when the same oligomer substrate is used, it is evident that, in control E. coli lacking Fpg and Nei, only endogenous Nth formed a major trapped complex (lane 3). Crude bacterial lysates harboring WT NEIL2 formed a trapped complex of the same size as the purified recombinant NEIL2 (lane 2) used as a marker. However, the lack of such trapped complexes with crude lysates expressing various mutant NEIL2 proteins (lanes 5–8) suggests that the mutants are inactive as AP lyases. This loss of activity is probably the result of a loss of the zinc finger motif, critical either for structural integrity or the DNA binding activity of this enzyme.DNA Binding and Incision Activity of Purified WT and Mutant NEIL2—Wild type and NEIL2 mutants (C291S, C315S, C318S, H295A, and R310Q) were purified to apparent homogeneity (Fig. 3). Surprisingly, the H295A mutant migrated abnormally and ran more slowly than the WT or the other mutants. We have shown earlier that NEIL2 is active in excising several cytosine-derived lesions and has robust activity for 5-OHU in bubble DNA (14Dou H. Mitra S. Hazra T.K. J. Biol. Chem. 2003; 278: 49679-49684Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Purified WT and mutant NEIL2 polypeptides were tested for DNA incision assays with a 5-OHU-containing bubble oligomer (5 OHU·B11). The expected βδ-elimination product, i.e. cleaved 5-OHU-containing strand with 3′-phosphate termini, was observed with the WT NEIL2 protein (14Dou H. Mitra S. Hazra T.K. J. Biol. Chem. 2003; 278: 49679-49684Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). However, neither mutant NEIL2 generated the oligomer fragment to a detectable extent (Fig. 4A). The WT and mutant proteins were also assayed for their DNA binding activity by gel mobility shift assay with 5 OHU containing B11 oligomer. Again the DNA protein complex was observed only with WT NEIL2 (lane 2) but not the mutant proteins (Fig. 4B, lanes 3–6).Fig. 3Purification of WT and mutant NEIL2. WT (lane 1), C291S, H295A, R310Q, C315S, and C318S mutant NEIL2 (lanes 3–7) proteins purified as described under “Experimental Procedures” were analyzed by 12% SDS-PAGE. Lane 2, size markers (Bio-Rad).View Large Image Figure ViewerDownload (PPT)Fig. 4DNA incision and binding activities of purified WT and mutant NEIL2. A, DNA incision assay. C291S, H295A, C315S, and C318S mutants (50 nm, lanes 3–6) and WT NEIL2 (lane 2) were incubated with 5′-32P-labeled 5-OHU-containing 11-nt bubble oligomers (250 nm), and the reaction products were separated by denaturing gel electrophoresis. S indicates substrate, and P indicates the position of products in the gel. B, DNA binding assay. 5′-32P-labeled 5-OHU-containing 11-mer bubble (0.2 pmol) was incubated with 5 ng each of purified WT (lane 2), C291S (lane 3), H295A (lane 4), C315S (lane 5), and C318S (lane 6) for gel mobility shift analysis in 6% polyacrylamide.View Large Image Figure ViewerDownload (PPT)Zinc Content of Wild-type and Mutant NEIL2—Purified WT and C318S and H295A mutant NEIL2 were analyzed for zinc content by inductively coupled plasma mass spectrometry. The WT NEIL2 protein contained 0.97 ± 0.086 mol zinc/mol protein. In contrast, C318S and H295A mutants contained <0.1 mol of zinc/mol protein (Table I).Table IAnalysis of zinc content in WT and mutant NEIL2 by ICP-MSNEIL2 proteinsMol of zinc/mol of NEIL2Wild type0.97 ± 0.086C318S0.15 ± 0.014H295A0.08 ± 0.009 Open table in a new tab Structural Alterations in H295A and C318S NEIL2 Mutants—The presence of zinc" @default.
• W2020678474 created "2016-06-24" @default.
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• W2020678474 date "2004-11-01" @default.
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• W2020678474 title "Identification of a Zinc Finger Domain in the Human NEIL2 (Nei-like-2) Protein" @default.
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• W2020678474 doi "https://doi.org/10.1074/jbc.m406224200" @default.
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