The 2,6-diamino-4-hydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase of Escherichia coli, which is coded for by the fpg gene, excises purine bases with ring-opened imidazoles. In addition to the DNA glycosylase activity, we report that the Fapy-DNA glycosylase of E. coli has an associated activity, resistant to EDTA, that nicks DNA at apurinic/apyrimidinic (AP) sites. The levels of Fapy-DNA glycosylase and AP-nicking activity were parallel in crude lysates of E. coli HB101 harboring different plasmids constructed from the fpg gene. The fpg gene is different from the xth, nth, and nfo genes of E. coli, whose gene products also cleave DNA at AP sites. The Fapy-DNA glycosylase was purified to electrophoretic homogeneity. During this purification, the Fapy-DNA glycosylase copurified with an AP-nicking activity using chromatographic separations based on ion-exchange, molecular weight exclusion, and hydrophobicity. The cleavage at AP sites by the Fapy-DNA glycosylase left a 5'-phosphomonoester nucleotide at one terminus. In addition, DNA containing reduced AP sites was not nicked by the Fapy-DNA glycosylase. These data suggest that the mechanism of cleavage involved beta elimination. Therefore, this activity of the Fapy-DNA glycosylase nicking DNA at AP sites should be referred to as an AP lyase. The 3' terminus did not prime nick-translation by E. coli DNA polymerase I. However, the 3' terminus becomes a substrate for nick-translation if first allowed to react with calf intestine phosphatase or the E. coli exonuclease III. These data suggest that the repair of the Fapy lesion at least to some extent results in the formation of both 5'- and 3'-phosphomonoester nucleotides and the release of the deoxyribose.

A cDNA plasmid expression library was constructed from the poly(A)+ mRNA of H4 cells, a rat hepatoma cell line. The library was introduced into Escherichia coli strain BH290 deficient in the repair of 3-methyladenine (3-meAde) residues in DNA. This DNA repair deficiency renders the stain phenotypically sensitive to treatment with alkylating agents. The cDNA library was screened for survivors to methylmethane sulfonate. BH290 cells hosting one of the plasmids, pAPDG10 (Alkylated N-Purine-DNA Glycosylase), from surviving cells had a sensitivity to MMS equivalent to that of the wild type strain. Crude extracts of BH290 cells harboring the pAPDG10 plasmid released 3-meAde and 7-methylguanine residues from DNA methylated with [methyl-3H]dimethylsulfate. The cDNA sequence of 993 bp inserted in pAPDG10 has a single open reading frame greater than 85 amino acids in length. The derived APDG protein sequence of 253 amino acids and the 3-meAde-DNA glycosylase II of E. coli coded for by the alkA gene have regions of conserved sequences. Analysis of the genomic DNA using Southern hybridization suggests that the APDG gene has a minimal size of 6.5-12 kb. Northern blot analysis shows that the transcript produced in H4 cells is also present in normal rat liver cells.

The formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase) of Escherichia coli (E. coli) was overexpressed by cloning the fpg+ gene on a multicopy plasmid and placing this gene under the control of the lac promoter. The lac promoter contributed significantly to the overall expression of the fpg gene only after the deletion of an inverted repeat sequence located immediately upstream from the fpg promoter. The biological purpose of the inverted repeat sequence may be associated with the termination of an adjacent gene transcribed in the same direction as the fpg gene in E. coli. Cells harboring the fpg gene under the control of the lac promoter were able to produce the Fapy-DNA glycosylase as at least 17% of the total soluble proteins. Such strains allow the preparation of milligram quantities of pure protein for use in the study of its catalytic properties and three dimensional crystal structure.

The structural and energetic consequences of cytosine methylation in the 5-position on the supercoil-dependent B-Z equilibrium in alternating dC-dG sequences cloned into recombinant plasmids were investigated. The helical parameters determined with the band shift method for right-handed [10.7 base pairs (bp)/turn] and left-handed (12.8 bp/turn) 5MedC-dG inserts were different from the helical repeat values for unmethylated dC-dG inserts (10.5 bp/turn in the right-handed and 11.5 bp/turn in the left-handed form). We analyzed the thermodynamic parameters delta GBZ (free energy difference per base pair between right-handed and left-handed helix structure), delta Gjx (free energy for formation of one B-Z junction), and b (helix unwinding at a junction region) for varying lengths of dC-dG inserts by two-dimensional gel electrophoresis and application of a statistical mechanics model. A comparison of plasmids fully methylated in vitro with HhaI methylase and their unmethylated counterparts revealed that delta Gjx is not significantly changed by cytosine methylation. However, this base modification results in an approximate 3-fold decrease of delta GBZ and an approximate 2-fold decrease of the unwinding b at B-Z junction regions. Analysis of a pair of related plasmids, each containing two dC-dG blocks, revealed qualitatively different transition behaviors. When the two dC-dG blocks were separated by 95 bp of a mixed sequence, they underwent independent B to Z transitions with separate nucleation events and junction formations. When the two blocks were separated by only a 4 bp GATC sequence, only one nucleation event was necessary, and the Z-helix spread across the nonalternating GATC region.(ABSTRACT TRUNCATED AT 250 WORDS)

The entropy and enthalpy changes which contribute to the thermodynamics of the B to Z transition were determined for three recombinant plasmids containing a (dC-dG)16 tract and for a plasmid containing a pair of (dT-dG)20 regions. For each base pair which adopts a left-handed conformation in the plasmids with (dC-dG)16 sequences, the delta HBZ and delta SBZ are -2.1 kcal/mol bp and -8.8 cal/K-mol bp, respectively. In the plasmid containing the (dT-dG)20 tracts, however, the delta HBZ and delta SBZ values are 0.58 kcal/mol bp and -0.76 cal/K-mol bp, respectively. Also, these determinations show that for each B-Z junction that forms in the plasmids containing the (dC-dG), the enthalpy and entropy changes are 24 kcal/mol junction and 65 cal/K-mol junction, whereas for the (dT-dG) plasmid, the enthalpy and entropy changes are -1.8 kcal/mol junction and -22 cal/K-mol junction, respectively. Those values for the enthalpy and entropy changes for the formation of a BZ junction in (dC-dG) and (dT-dG) plasmids suggest that the properties and possibly the structures of the junctions are different. Calculations using the enthalpy and entropy changes determined in this study reveal that the B to Z transition in plasmids containing (dC-dG) blocks are more temperature-dependent than the transitions in plasmids with (dT-dG) blocks. Surprisingly, at temperatures above 60 degrees C, calculations indicate that the B to Z transitions in (dT-dG) plasmids should be energetically favored over that transition in (dC-dG) plasmids.

An Escherichia coli genomic library composed of large DNA fragments (10-15 kb) was constructed using the plasmid pBR322 as vector. From it 700 clones were individually screened for increased excision of the ring-opened form of N7-methylguanine (2-6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine) or Fapy. One clone overproduced the Fapy-DNA glycosylase activity by a factor of 10-fold as compared with the wild-type strain. The Fapy-DNA glycosylase overproducer character was associated with a 15-kb recombinant plasmid (pFPG10). After subcloning a 1.4-kb fragment which contained the Fapy-DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively. The cells harbouring pFPG60 displayed a 50- to 100-fold increase in glycosylase activity and overexpressed a 31-kd protein. From these cells the Fapy-DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 31 kd on SDS-polyacrylamide gels. The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy-DNA glycosylase. The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol. wt of 30.2 kd.

Homogeneous Fpg protein of Escherichia coli has DNA glycosylase activity which excises some purine bases with damaged imidazole rings, and an activity excising deoxyribose (dR) from DNA at abasic (AP) sites leaving a gap bordered by 5'- and 3'-phosphoryl groups. In addition to these two reported activities, we show that the Fpg protein also catalyzes the excision of 5'-terminal deoxyribose phosphate (dRp) from DNA, which is the principal product formed by the incision of AP endonucleases at abasic sites. Moreover, the rate of the Fpg protein catalysis for the 2,6-diamino-4-hydroxy-5-formamidopyrimidine-DNA glycosylase activity is slower than the activities excising dR from abasic sites and dRp from abasic sites preincised by endonucleases. The product released by the Fpg protein in the excision of 5'-terminal dRp from an abasic site preincised by an AP endonuclease is a single base-free unsaturated dRp, suggesting that the excision results from beta-elimination. The release of 5'-terminal dRp by crude extracts of E. coli from wild type and fpg-mutant strains shows that the Fpg protein is one of the major EDTA-resistant activities catalyzing this reaction.

Purpose

1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-cyanuric acid (cyanuric acid nucleoside or dCa) has been shown to be formed upon exposure of 8-oxo-7,8-dihydroguanine- (8-oxoG) containing oligodeoxyribonucleotides (ODN) to oxidizing agents. When present in DNA, cyanuric acid (Ca) is readily bypassed by Escherichia coli DNA polymerases, which preferentially incorporate 2'-deoxyadenosine-5'-monophosphate (dAMP) opposite to the lesion. Therefore, Ca could be a mutagenic DNA lesion yielding G.C to T.A transversions like 8-oxoG. These results call attention to the potential importance of secondary oxidation products of 8-oxoG. The present study investigates the capability of several DNA N-glycosylases to remove the Ca lesion in DNA.

Materials and methods

A site-specifically modified 22-mer ODN containing a single Ca residue was hybridized with complementary sequences yielding four DNA duplexes harbouring Ca opposite each of the regular DNA bases. The four Ca.N duplexes were used as substrates for nine DNA N-glycosylases from bacterial, yeast or human origin.

Results

The results show that the human methylpurine DNA N-glycosylase (Mpg) can remove Ca from DNA duplexes. Interestingly, oxidized base-specific DNA N-glycosylases, Fpg, Nth, Ntg1, Ntg2, Ogg1, hNth1 and hOgg1, cannot repair Ca in DNA. Furthermore, the removal of Ca by Mpg varied markedly depending on the opposite DNA base, the rank being Ca.C=Ca.T>Ca.G=Ca.A.

Conclusions

8-OxoG-derived lesions in DNA such as spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), oxaluric acid (Oa), oxazolone (Oz) and Ca are substrates of base excision repair DNA N-glycosylases. Most of them, Sp, Gh, Oa and Oz, are substrates of the oxidized bases-specific enzymes such as Nth or Fpg. In contrast, Ca is substrate of the human methylpurine DNA N-glycosylase (Mpg).

The repair of 2,6-diamino-4-hydroxy-5-N-methyl-formamidopyrimidine (Fapy) residues in DNA is performed by a Fapy-DNA glycosylase activity which is encoded for by the fpg gene in Escherichia coli. Besides DNA glycosylase activity, this protein, the FPG protein, is endowed with an EDTA-resistant activity nicking DNA at apurinic/apyrimidinic (AP) sites. To overproduce the FPG protein, the fpg gene was placed under the control of the tac promoter in the expression vector pKK223-3 yielding the pFPG230 plasmid. The production of the FPG protein in cells harboring the pFPG230 plasmid was 800-fold higher than that of the wild type strain after induction by isopropyl-beta-D-thio-galactopyranoside. From these cells, the FPG protein was purified to homogeneity in sufficient quantity to study its physical and catalytic properties. In its active form, the FPG protein is a globular monomer of 31 kDa and has an experimentally measured isoelectric point of 8.5. When the FPG protein is heat-denatured in the presence of EDTA the two activities are more rapidly inactivated than when heated in the absence of EDTA, suggesting that the FPG protein possesses a tightly bound metal ion. Atomic absorption spectrophotometric analysis shows that there is one zinc/FPG protein molecule. The FPG protein is different from previously described DNA glycosylases and AP-nicking enzymes in E. coli. The contribution of the AP-nicking activity associated with the FPG protein represents 10-20% of the total EDTA-resistant AP-nicking activities in E. coli.