Role of the N-terminal Proline Residue in the Catalytic Activities of the Escherichia coli Fpg Protein

The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 → Gly (FpgP2G), Pro-2 → Thr (FpgP2T), and Pro-2 → Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C → T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitroand in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity. The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 → Gly (FpgP2G), Pro-2 → Thr (FpgP2T), and Pro-2 → Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C → T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitroand in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity. 7,8-dihydro-8-oxoguanine 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine apurinic/apyrimidinic, abasic polyacrylamide gel electrophoresis 7,8-Dihydro-8-oxoguanine (8-oxoG)1 is the major mutagenic base lesion generated in DNA by active oxygen during normal metabolism (1.Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4474) Google Scholar). Because 8-oxoG pairs preferentially with adenine rather than cytosine, it generates transversion mutations after replication (2.Grollman A. Trends Genet. 1993; 9: 246-249Abstract Full Text PDF PubMed Scopus (748) Google Scholar). To maintain the genetic integrity, this oxydized base is repaired, in Escherichia coli, by the Fpg protein (2.Grollman A. Trends Genet. 1993; 9: 246-249Abstract Full Text PDF PubMed Scopus (748) Google Scholar, 3.Tchou J. Kasai H. Shibutani S. Chung M.-H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1993; 88: 4690-4694Crossref Scopus (708) Google Scholar) coded for by the fpg gene (4.Boiteux S. O'Connor T. Laval J. EMBO J. 1987; 6: 3177-3183Crossref PubMed Scopus (260) Google Scholar). The physical and enzymatic properties of the protein have been established (5.Laval J. Boiteux S. O'Connor T.R. Mutat. Res. 1990; 233: 73-79Crossref PubMed Scopus (41) Google Scholar). It is a globular monomer of 30.2 kDa (269 amino acids), pI = 8.6, that contains one zinc atom/protein molecule present in a zinc finger motif (5.Laval J. Boiteux S. O'Connor T.R. Mutat. Res. 1990; 233: 73-79Crossref PubMed Scopus (41) Google Scholar). In vitro, the Fpg protein exhibits three catalytic activities: (i) it is a DNA glycosylase that liberates modified residues such as 2,6-diamino-4-hydroxy-5-N-methyl-formamido pyrimidine (Fapy) (6.Chetsanga C.J. Lindahl T. Nucleic Acids Res. 1979; 6: 3673-3683Crossref PubMed Scopus (238) Google Scholar), 8-oxoG (3.Tchou J. Kasai H. Shibutani S. Chung M.-H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1993; 88: 4690-4694Crossref Scopus (708) Google Scholar, 7.Boiteux S. Gajewski E. Laval J. Dizdaroglu M. Biochemistry. 1992; 31: 106-110Crossref PubMed Scopus (581) Google Scholar), 8-oxoA, the imidazole ring-opened forms of adenine and guanine (7.Boiteux S. Gajewski E. Laval J. Dizdaroglu M. Biochemistry. 1992; 31: 106-110Crossref PubMed Scopus (581) Google Scholar), and modified pyrimidines, such as 5-hydroxycytosine, 5-hydroxyuracil, β-ureidoisobutiric acid, α-R-hydroxy-β-ureidoisobutiric acid (8.Jurado J. Saparbaev M. Matray M.J. Greenberg M.M. Laval J. Biochemistry. 1998; 37: 7757-7763Crossref PubMed Scopus (63) Google Scholar) (for review, see Ref. 9.Laval J. Jurado J. Saparbaev M. Sidorkina O. Mutat. Res. 1998; 402: 93-102Crossref PubMed Scopus (87) Google Scholar), (ii) it is an AP lyase that incises DNA at abasic sites by a β-δ-elimination mechanism (10.O'Connor T.R. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5222-5226Crossref PubMed Scopus (149) Google Scholar, 11.Bailly V. Verly W.G. O'Connor T. Laval J. Biochem. J. 1989; 262: 581-589Crossref PubMed Scopus (148) Google Scholar), and (iii) it is a deoxyribophosphodiesterase that removes 5′-terminal deoxyribose phosphate residues (12.Graves R.J. Felzenszwalb J. Laval J. O'Connor T. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar). Therefore the Fpg protein belongs to the class of DNA glycosylases endowed with a β-lyase activity (9.Laval J. Jurado J. Saparbaev M. Sidorkina O. Mutat. Res. 1998; 402: 93-102Crossref PubMed Scopus (87) Google Scholar). It was hypothesized that the glycosylase and lyase functions of DNA glycosylases/AP lyases are mechanistically coupled (13.Kow Y.W. Wallace S.S. Biochemistry. 1987; 26: 8200-8208Crossref PubMed Scopus (160) Google Scholar, 14.Weiss B. Grossman L. Adv. Enzymol. 1987; 60: 1-34PubMed Google Scholar, 15.Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar). A mechanism involving the nucleophilic attack on C1′ of the modified deoxynucleoside targeted for excision has been proposed (13.Kow Y.W. Wallace S.S. Biochemistry. 1987; 26: 8200-8208Crossref PubMed Scopus (160) Google Scholar, 14.Weiss B. Grossman L. Adv. Enzymol. 1987; 60: 1-34PubMed Google Scholar, 15.Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar) to explain the catalytic action of this class of enzymes. In this mechanism, the catalytic nucleophile attacks the glycosidic bond, displacing the aberrant base and forming a transient intermediate (Schiff base) with the C1′-deoxyribose moiety. The isomerization of this transient complex leads to cleavage of the AP site through a conjugate elimination mechanism. It has been proposed that most DNA glycosylases/AP lyases use the ε- NH2 group of a lysine as the catalytic nucleophile (16.Nash H.M. Lu R. Lane W.S. Verdine G.L. Chem. Biol. 1997; 4: 693-702Abstract Full Text PDF PubMed Scopus (164) Google Scholar). For the Nth, hOgg1, and hNth proteins, the lysine residues at positions 120, 249, and 212, respectively, are believed to be the catalytic nucleophiles (16.Nash H.M. Lu R. Lane W.S. Verdine G.L. Chem. Biol. 1997; 4: 693-702Abstract Full Text PDF PubMed Scopus (164) Google Scholar, 17.Thayer M.M. Ahern H. Xing D. Cunningham R.P. Tainer J.A. EMBO J. 1995; 14: 4108-4120Crossref PubMed Scopus (439) Google Scholar, 18.Ikeda 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 (224) Google Scholar). However, the NH2 group of the N-terminal amino acid can be the catalytic amine, as shown in the case of the bacteriophage T4 endonuclease V (19.Dodson M.L. Schrock III, R.D. Lloyd R.S. Biochemistry. 1993; 32: 8284-8290Crossref PubMed Scopus (137) Google Scholar). For the Fpg protein, the N-terminal proline residue was shown to be linked, in a Schiff base complex, with DNA containing 8-oxoG residues. It was suggested that this proline was the nucleophile that initiates the catalytic excision of 8-oxoG (20.Zharkov D.O. Reiger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-53413Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The N-terminal formylmethionine of the cloned wild-type Fpg protein is post-translationally cleaved in E. coli (4.Boiteux S. O'Connor T. Laval J. EMBO J. 1987; 6: 3177-3183Crossref PubMed Scopus (260) Google Scholar). However, because in some mutant Fpg proteins, the formylmethionine could not be cleaved, the amino acid sequence of the Fpg protein was numbered Met-1, Pro-2, etc. (21.Sidorkina O.M. Laval J. Nucleic Acids Res. 1998; 26: 5351-5357Crossref PubMed Scopus (39) Google Scholar, 22.Rabow L.E. Kow Y.W. Biochemistry. 1997; 36: 5084-5096Crossref PubMed Scopus (76) Google Scholar). To further understand the role of the N-terminal proline, in the various catalytic activities of the Fpg protein, Pro-2 was mutated to three different amino acids: Gly, Thr, or Glu generating the FpgP2G, FpgP2T, and FpgP2E mutant proteins, respectively. These proteins were purified, and their catalytic properties toward several substrates as well as their binding constants were established. Finally, we have determined the in vivo properties of the mutant Fpg proteins. Our results show that Pro-2 is involved in the various enzymatic activities of the Fpg protein, although at different extent, and that one can observe the formation of a robust Schiff base intermediate with an oligonucleotide containing an abasic site despite the lack of a detectable AP lyase activity. The following strains, derived from the E. coli K12 strain, were used: CC 104 (araΔ (gpt-lac)5, rpsL/F′ (lacI378,lacZ461, proA+B+] (23.Cupples C.G. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5345-5349Crossref PubMed Scopus (389) Google Scholar), BH 990 (as CC 104 but fpg::kanR,mutY::kanR) (24.Duwat P. de Oliveira R. Ehrlich S.D. Boiteux S. Microbiology. 1995; 141: 411-417Crossref PubMed Scopus (58) Google Scholar), BH 20 (as AB 1157 but fpg::kanR) (25.Boiteux S. Huisman O. Mol. Gen. Genet. 1989; 215: PubMed Scopus (76) Google Scholar), and J. T. A Scholar). The containing the cloned (4.Boiteux S. O'Connor T. Laval J. EMBO J. 1987; 6: 3177-3183Crossref PubMed Scopus (260) Google Scholar), T.R. Boiteux S. Laval J. 1989; Google Scholar), and were from DNA DNA and DNA were from DNA were from and respectively, and as by the was from were from and were from The substrates and E. coli DNA containing abasic sites were as (4.Boiteux S. O'Connor T. Laval J. EMBO J. 1987; 6: 3177-3183Crossref PubMed Scopus (260) Google Scholar, T.R. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5222-5226Crossref PubMed Scopus (149) Google Scholar). The of gel DNA and were as by J. T. A Scholar) or by the The was for the construction of mutated fpg The was by and DNA The to amino acid of the Fpg protein are in and were from To an to the of the the was by a that was and and cloned was to the mutant Fpg proteins with amino acid Pro-2 → Gly, Pro-2 → and Pro-2 → The are and The DNA sequence was by the with DNA To further the mutant proteins, the sequence of the N-terminal amino of protein was determined not of sequence mutant fpg gene → → → 8-oxoG-DNA glycosylase activity activity DNA binding the mutant in a The the mutant The to the Fpg mutant proteins, of or 8-oxoG-DNA glycosylase activity of DNA at AP and formation of in the of have been (21.Sidorkina O.M. Laval J. Nucleic Acids Res. 1998; 26: 5351-5357Crossref PubMed Scopus (39) Google Scholar). For DNA binding the gel duplex containing a abasic site were as B. Boiteux S. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar). The BH990 (fpg mutY) was by or The was containing (4.Boiteux S. O'Connor T. Laval J. EMBO J. 1987; 6: 3177-3183Crossref PubMed Scopus (260) Google Scholar, J. Boiteux S. O'Connor T.R. Mutat. Res. 1990; 233: 73-79Crossref PubMed Scopus (41) Google Scholar, C.J. Lindahl T. Nucleic Acids Res. 1979; 6: 3673-3683Crossref PubMed Scopus (238) Google Scholar) were at in of containing and containing and at a of The spontaneous in the was for the of (21.Sidorkina O.M. Laval J. Nucleic Acids Res. 1998; 26: 5351-5357Crossref PubMed Scopus (39) Google Scholar, P. de Oliveira R. Ehrlich S.D. Boiteux S. Microbiology. 1995; 141: 411-417Crossref PubMed Scopus (58) Google Scholar). have various E. coli mutant Fpg proteins to the of the N-terminal proline in the various enzymatic activities of this protein. was mutated to Gly, Thr, or Gly and Thr were for because are amino with and and D. Trends Sci. 1990; Full Text PDF PubMed Scopus Google Scholar). Because in the wild-type Fpg protein, the of N-terminal is we examined the of an when Pro-2 is to Glu and The mutant proteins were in E. coli (fpg to by the wild-type The of mutant was comparable with that of as by the of the protein in not In of E. coli the mutant FpgP2G and FpgP2T, a glycosylase activity was but to a than in the wild-type protein. glycosylase activity could be in of coli the Fpg mutant In when DNA was as a barely detectable AP activity was in all the in the of as the and was to Nth, or proteins present in the glycosylase activity and AP lyase activity in of E. coli for wild-type and mutant Fpg of of glycosylase activity and activity DNA at AP sites were in as as substrates and E. coli DNA, respectively. For glycosylase one is as the of the protein that of in at For the activity DNA at AP sites one is as the of protein that of acid oligonucleotide in at in a The glycosylase activity and activity DNA at AP sites were in as as substrates and E. coli DNA, respectively. For glycosylase one is as the of the protein that of in at For the activity DNA at AP sites one is as the of protein that of acid oligonucleotide in at of the wild-type and mutant the after were than of not and were to the various enzymatic properties of the mutant Fpg proteins. To further the N-terminal amino were Because the of the cleavage of the formylmethionine the is determined by the amino acid to the formylmethionine in the protein B. H. J. Biochem. PubMed Scopus Google Scholar), it was that such cleavage not in the mutant enzymes. In the N-terminal was as shown by in the FpgP2G and FpgP2T proteins. in the the formylmethionine was present not of the various enzymatic properties of the Fpg protein were detectable the FpgP2E mutant protein not The of FpgP2G and to residues was The of the activity of the Fpg wild-type and mutant proteins that FpgP2G and FpgP2T had a comparable but 10% of glycosylase activity The of Fpg mutant proteins to 8-oxoG was as a oligonucleotide containing a 8-oxoG shown in the of of the duplex was for the or mutants as with the wild-type Fpg protein It was that the excision of 8-oxoG residues was not following the action of the in the or of 10% DNA at AP the of of the was not These that the N-terminal proline is mandatory for 8-oxoG but less for residues The of and mutant proteins to cleave abasic sites was different In the coli DNA containing AP sites was as substrate. In this the Fpg protein at AP sites and liberates the mutant Fpg proteins, detectable AP activity was for of the mutant proteins In the a oligonucleotide with an preformed abasic site at a was as The mutant had less than of the wild-type activity generating not that the N-terminal proline is mandatory for the AP lyase activity when upon preformed AP The Fpg protein forms a transient Schiff base intermediate with 8-oxoG/C containing DNA in a J. Grollman A.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). after with or is to a Fpg complex J. Grollman A.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, B. M.L. Lloyd R.S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). have the of the mutant FpgP2G and FpgP2T proteins to 8-oxoG/C DNA in the of The of FpgP2G and FpgP2T was is in with the barely detectable 8-oxoG-DNA glycosylase activity In when the AP site containing DNA is as the mutant a Schiff base intermediate with an than that of the wild-type protein However, FpgP2G has an AP lyase activity upon preformed AP site These results show that it is to a robust Schiff base intermediate an an DNA AP lyase activity. The mutant FpgP2G and proteins were for their to a duplex DNA containing a tetrahydrofuran a B. M.L. Lloyd R.S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. J. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). The constants of mutant FpgP2G and were and respectively, comparable with that for wild-type Fpg protein These results that the of the N-terminal proline for or acid not with DNA constants for wild-type and mutant and Fpg were by gel duplex containing a tetrahydrofuran For see Ref. B. Boiteux S. Nucleic Acids Res. 1992; PubMed Scopus Google The of from for protein were less in a The were by gel duplex containing a tetrahydrofuran For see Ref. B. Boiteux S. Nucleic Acids Res. 1992; PubMed Scopus Google The of from for protein were less E. DNA glycosylase activities that spontaneous by the Fpg protein, 8-oxoG residues in DNA (3.Tchou J. Kasai H. Shibutani S. Chung M.-H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1993; 88: 4690-4694Crossref Scopus (708) Google Scholar, 7.Boiteux S. Gajewski E. Laval J. Dizdaroglu M. Biochemistry. 1992; 31: 106-110Crossref PubMed Scopus (581) Google Scholar), and the protein, the adenine residues 8-oxoG S. Miller J.H. P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: Scholar, Grollman A.P. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, J. Grollman A.P. Miller J.H. Biochemistry. 1992; 31: PubMed Scopus Google Scholar). of and of E. coli mutY) results in a G/C → T/A spontaneous (24.Duwat P. de Oliveira R. Ehrlich S.D. Boiteux S. Microbiology. 1995; 141: 411-417Crossref PubMed Scopus (58) Google Scholar, Grollman A.P. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). To the of the active FpgP2G and FpgP2T proteins in we have the spontaneous in the mutY) for the wild-type or mutant Fpg proteins The BH990 (fpg mutY) a in G/C → T/A transversion with the The spontaneous because of fpg and is completely by of the for the wild-type Fpg protein. In the for FpgP2G protein barely and the FpgP2T protein not the spontaneous of the E. coli mutant the role of N-terminal proline for the activity of this protein, the of the mutagenic 8-oxoG of E. coli and its BH990 (fpg mutY) the or mutant 104 990 (fpg mutY) = = = = = = = of E. coli CC 104 or BH 990 (fpg mutY) with or the fpg gene or mutant were For see in a of E. coli CC 104 or BH 990 (fpg mutY) with or the fpg gene or mutant were For see It has been proposed that the N-terminal proline of the Fpg protein could be the nucleophile involved in the Schiff base formation with 8-oxoG residues (20.Zharkov D.O. Reiger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-53413Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). To in the of this amino acid upon the various activities of the Fpg protein, we have three mutated proteins at this by FpgP2G, FpgP2T, and These mutated proteins were to and for their various enzymatic The results that the of the N-terminal proline barely the DNA binding of the mutated proteins as with the These are in with the results of and Grollman J. Grollman A.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), that the of a protein at the of Fpg protein not DNA as a DNA containing residues, the FpgP2G and FpgP2T proteins were active 10% of the activity of the wild-type The of the 8-oxoG-DNA glycosylase activity of the mutated proteins that the activity of the FpgP2G and FpgP2T mutants is and that mutants barely Schiff base with containing 8-oxoG residues. results that the N-terminal proline is for the of the 8-oxoG residue It has been proposed that 8-oxoG is the by the Fpg protein in vivo to G/C → T/A transversion (1.Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4474) Google Scholar, A. Trends Genet. 1993; 9: 246-249Abstract Full Text PDF PubMed Scopus (748) Google Scholar, 9.Laval J. Jurado J. Saparbaev M. Sidorkina O. Mutat. Res. 1998; 402: 93-102Crossref PubMed Scopus (87) Google Scholar, O.M. Laval J. Nucleic Acids Res. 1998; 26: 5351-5357Crossref PubMed Scopus (39) Google Scholar). The results show that the FpgP2G at and that the FpgP2T mutant not the spontaneous G/C → T/A in vivo results are in with the minute 8-oxoG-DNA glycosylase activity of the proteins, Therefore the and excision of 8-oxoG residues are complex because at amino and are involved in (21.Sidorkina O.M. Laval J. Nucleic Acids Res. 1998; 26: 5351-5357Crossref PubMed Scopus (39) Google Scholar, 22.Rabow L.E. Kow Y.W. Biochemistry. 1997; 36: 5084-5096Crossref PubMed Scopus (76) Google Scholar). The N-terminal proline is for the β-lyase activity of the Fpg protein at preformed AP different substrates containing preformed AP site were of the mutant proteins had detectable activity upon The mechanism of action of the has been proposed to a Schiff base intermediate that can be by of the complex with a (13.Kow Y.W. Wallace S.S. Biochemistry. 1987; 26: 8200-8208Crossref PubMed Scopus (160) Google Scholar, 14.Weiss B. Grossman L. Adv. Enzymol. 1987; 60: 1-34PubMed Google Scholar, 15.Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar). In an of a of DNA activity with their substrates has been 15.Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar, D.O. Reiger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-53413Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, O.M. Laval J. Nucleic Acids Res. 1998; 26: 5351-5357Crossref PubMed Scopus (39) Google Scholar, 22.Rabow L.E. Kow Y.W. Biochemistry. 1997; 36: 5084-5096Crossref PubMed Scopus (76) Google Scholar, J. Grollman A.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, B. M.L. Lloyd R.S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, R. H.M. Verdine G.L. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, and M.J. Jurado J. E. Laval Nucleic Acids Res. 1998; Scopus Google and this The results 8-oxoG containing oligonucleotide this by the wild-type Fpg protein. It 8-oxoG residues and readily Schiff base the FpgP2G or FpgP2T proteins a 8-oxoG-DNA glycosylase activity barely Schiff base with 8-oxoG/C DNA. when the oligonucleotide containing a preformed abasic site and FpgP2G, a mutant protein of activity the formation and the of the complex in the of was comparable with the of complex with the wild-type Fpg protein. that it is to the formation of the complex from the of the DNA at abasic The of the N-terminal proline residue an of a readily to its its the Fpg mutant produced the Schiff base intermediate with preformed AP site but did not AP The E. coli protein is a DNA glycosylase adenine residues in a S.D. S.S. Nucleic Acids Res. 1998; 26: PubMed Scopus Google Scholar, D.O. Grollman A.P. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). at than forms a intermediate with its involving D.O. Grollman A.P. Biochemistry. 1998; 37: PubMed Scopus Google Scholar) but has or AP lyase activity S.D. S.S. Nucleic Acids Res. 1998; 26: PubMed Scopus Google Scholar, D.O. Grollman A.P. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). It has been shown that the amino acid to generates a protein its DNA glycosylase activity but DNA at abasic site by a elimination mechanism the formation of a Schiff base intermediate J. J. Lu J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mutant protein has the properties of the Fpg These that it is to the formation of the Schiff base complex from the of the DNA at abasic The results the different role of the N-terminal proline of the Fpg protein in the of the different However, it is not that the amino acid could the the role of some amino in the active site of the protein. For we have shown that the in the FpgP2G mutant did not the of this protein as determined by the and the of Sidorkina O.M. Jurado J. M. P. Laval J. R. J. Biochem. 1998; PubMed Scopus Google Scholar). However, were in the of the mutant at 20 that the residues are as with the wild-type protein. The FpgP2E protein is of enzymatic activity but has a binding to the FpgP2G are in the of FpgP2E at 20 at that its at 20 is the as at wild-type FpgP2G, and such The lack of the at the of the of the protein could explain the lack of enzymatic activity of the Fpg protein. J. and J. for the of oligonucleotide containing the tetrahydrofuran residue and for and J. for protein

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