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Originally published In Press as doi:10.1074/jbc.M006125200 on August 23, 2000

J. Biol. Chem., Vol. 275, Issue 45, 35471-35477, November 10, 2000
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Identification of Repair Enzymes for 5-Formyluracil in DNA

Nth, Nei, AND MutM PROTEINS OF ESCHERICHIA COLI*

Qiu-Mei ZhangDagger , Izumi MiyabeDagger , Yukiko MatsumotoDagger , Katsuhito Kino§, Hiroshi Sugiyama§, and Shuji YoneiDagger

From the Dagger  Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan and the § Institute for Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Received for publication, July 12, 2000, and in revised form, August 7, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood. In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites. The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides. Extracts from strains defective in the nth, nei, or mutM gene lacked one of the proteins. All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant. The introduction of a plasmid carrying the nth, nei, or mutM gene into the E. coli triple mutant restored the formation of the corresponding protein-DNA complex. Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH4. Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site. In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E. coli. The mutation frequency of the plasmid was significantly increased in the E. coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains. From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reactive oxygen species are generated in living cells during normal cellular metabolism and by exogenous sources such as ionizing radiation and various chemical oxidants (1, 2). Reactive oxygen species cause damage to DNA, producing a wide variety of oxidative modifications to purines and pyrimidines (3-7). Unrepaired oxidative DNA damage has been suggested to play a role in cancer, aging, and many degenerative pathologies in humans (1, 8-10). In most organisms, the repair of oxidatively damaged bases in DNA is primarily mediated by the base excision repair pathways (5, 6, 11-13).

The 5,6-double bond of pyrimidines is vulnerable to hydroxyl radicals generated by ionizing radiation and oxidizing agents. From thymine, 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), 5-hydroxy-5,6-dihydrothymine and a number of ring contraction and fragmentation products are formed (5, 6, 11-13). Although none of these lesions are strongly mutagenic, thymine glycol and urea in the template block DNA synthesis in vitro (5, 6, 14). These products are primarily recognized by endonuclease III (Nth) and endonuclease VIII (Nei), encoded by the nth and nei genes, respectively (15, 16), in Escherichia coli (5, 6, 17-19). These enzymes are DNA glycosylases/AP1 lyases that catalyze both the cleavage of the glycosylic bond to release damaged bases and the incision of the phosphodiester backbone at the resulting AP site via beta  or beta - and delta -elimination reactions (5, 6, 11-13, 20-23). The exocyclic methyl group of thymine does not escape oxidative damage. When cellular DNA is exposed to ionizing radiation and oxidizing agents, several thymine hydroperoxides are formed (7, 24, 25). 5-Hydroperoxymethyluracil spontaneously decompose to form 5-formyluracil (5-foU) and 5-hydroxymethyluracil (7, 24, 25). Several reports have shown that 5-foU is a potentially mutagenic lesion. Kasai et al. (26) found that 5-formyl-2'-deoxyuridine is mutagenic to Salmonella typhimurium TA102 when added to the culture medium. Recently, Fujikawa et al. (27) also reported that 5-formyl-deoxyUTP added to the culture medium causes mutations in E. coli. In addition, 5-foU directs misincorporation of mismatched bases opposite the lesion during DNA synthesis in vitro (28-30).

Bjelland et al. (31) and Masaoka et al. (32) have reported that E. coli AlkA protein (3-methyladenine DNA glycosylase II) removes 5-foU from DNA in vitro. Mammalian cells have been shown to possess DNA glycosylase activities that remove the 5-foU from DNA (33, 34). However, the human N-alkylpurine glycosylase that has a certain degree of overlapping substrate specificity with E. coli AlkA (6) does not show such repair activity for 5-foU (33). These facts suggest that the 5-foU-DNA glycosylase activity (33, 34) represents an enzyme different from previously characterized DNA glycosylases. These observations led us to examine whether E. coli has another DNA glycosylase(s) that recognize and remove 5-foU from DNA.

DNA glycosylases/AP lyases remove the modified base by breaking the glycosylic bond via formation of a Schiff base intermediate with the N-terminal NH2, imino group of proline or the NH2 group of an internal lysine residue, followed by a beta - (or beta - and delta -) elimination reaction (5, 12, 35-40). The transient covalent intermediate forms a stable product after its reduction by NaBH4 (35-40). The stable DNA-protein complex is called a "trapped complex." Detection of the trapped complex is useful for identifying DNA glycosylases accompanying AP lyase activity. In this study, we used the NaBH4 trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites (28, 29, 41). Here we report that E. coli possesses three kinds of DNA glycosylase/AP lyase activities acting on 5-foU, Nth, Nei, and MutM proteins.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacteria, Plasmids, and Media-- Bacterial strains used in this study were derivatives of E. coli K12. KSR1, KSR2, KSR3, KSR4, KSR5, KSR6, and KSR7, nth::Cm, nei::Kan, mutM::Tet, nth::Cm nei::Kan, nth::Cm mutM::Tet, nei::Kan mutM::Tet, nth::Cm nei::Kan mutM::Tet derivatives of CSH26 (42), respectively, were constructed using P1 transduction according to Miller (42) with a slight modification. BL21 (dcm ompT hsdS (rB-mB-) gal) and HB101 (leuB6 proA2 recA13 thi-1 ara-14 lacY1 galK2 xyl-5 mtl-1 rpsL20 supE44 hds20) were also used. MS23 was an alkA derivative of AB1157 (thr-1 ara-14 leuB6 lacY1 Delta (gpt-proA2)62 tsx-33 supE44 galK2 rac- hisG4 rfbD mgl-51 rpsL31 kdgK51 xyl-5 mtl-1 argE3 thi-1). KSR8 was nth::Cm nei::Kan mutM::Tet derivative of MS23. Other strains used and their relevant genotypes were: 21336, uvrD260; BW313, ung-1 dut-1; RPC500, nfo-1; JC7623, recB21 recC22 sbcB15; WD8014, mutD5; SYT5, mutT; CC104-1, mutL; BMH71-18, mutS215; CSH117, mutY; CS101, recA269; N3055, uvrA277; MG1655, dps; RP4182, Delta (dcm vsr); C218, Delta (ada25 alkB) ogt-1; GW3773, mutH471; RPC501, nfo-1 Delta (xthA). Plasmids pACYCnth and pACYCnei (43) were kind gifts from Dr. K. Yamamoto (Tohoku University).

LB broth (42) was used for culturing bacteria throughout this study. LB agar plates contained 2% agar. When necessary, ampicillin (50 µg/ml), kanamycin (50 µg/ml), tetracycline (25 µg/ml), or chloramphenicol (30 µg/ml) was added to the medium.

Enzymes and Chemicals-- Tetracycline hydrochloride, kanamycin, chloramphenicol, phenylmethylsulfonyl fluoride (PMSF), and NaBH4 were purchased from Wako Pure Chemicals. Ampicillin was obtained from Meiji Seika. T4 polynucleotide kinase was obtained from New England Biolabs. Plasmids pGEX-4T-1, pKK223-3, and pSVK3, glutathione-Sepharose 4B, thrombin, and prepacked columns for fast protein liquid chromatography, HiTrap Q, HiTrap SP, HiTrap Heparin, HiLoad Superdex 75, and Mono S were purchased from Amersham Pharmacia Biotech. Restriction enzymes, Taq DNA polymerase and T4 DNA ligase were obtained from Takara Shuzo and TOYOBO. Sequencing primers and BigDye Terminator Cycle Sequencing Kits were obtained from PerkinElmer Life Sciences. [gamma -32P]ATP (>148 TBq/mmol) was obtained from ICN Biomedicals Inc.

Preparation of Crude Extracts from E. coli Cells-- Overnight cultures of E. coli (50 ml) were centrifuged, and the pellets were resuspended in 3 ml of 50 mM Tris-HCl (pH 8.0) containing 1 mM EDTA and stored at -80 °C. The cells were sonicated in an ice water bath and then centrifuged at 11,000 × g for 30 min and subsequently at 15,000 × g for 30 min at 4 °C. The supernatants were stored at -80 °C.

Construction of Expression Plasmids of Nth, Nei, and MutM Proteins-- pACYCnth (43) was used as a template for PCR to amplify the nth gene. One primer (Pmet-1) contained a BamHI site followed by the sequence around the putative start codon (5'-TAGGGATCCCGTCTGATGAATAAAGCAA-3'), and the other (Pter-1) contained an EcoRI site followed by the sequence around the stop codon (5'-CCCGCGAATTCACCATGTCAAC-3'). PCR was performed in a reaction mixture containing 20 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM of the primer, and 5 units of Taq DNA polymerase. The amplified fragment was digested with BamHI and EcoRI, and then the BamHI/EcoRI fragment containing the whole coding region of the nth gene was subcloned into BamHI/EcoRI-digested pGEX-4T-1. The resulting plasmid was designated pGEX-Nth.

The DNA sequence encoding the whole open reading frame of the mutM gene was amplified by PCR from lambda 2A6 DNA (44) using the following primers: Pmet-2 (5'-TAATGAATTCGGAGATGCTATGCCTGAA-3') and Pter-2 (5'-TAATTAAGC TTAGCGTAGCGTTTATGCC-3'). PCR was performed as described above. The PCR product was subcloned into the plasmid pKK223-3 at the EcoRI/HindIII site. The recombinant plasmid was designated pKK-MutM.

pACYCnei (43) was used as a template for PCR to amplify the whole open reading frame of the nei gene. Two PCR primers were used: Pmet-3 (5'-ATGG AATTCACCATGCCTGAAGGC-3') and Pter-3 (5'-TAATAAGCTTGCTGACAGG GCCTG-3'). The PCR product was subcloned into the pKK223-3 at HindIII/EcoRI sites. The resulting plasmid was designated pKK-Nei. The sequences were checked to verify that no mutations had been introduced by the PCR.

Expression and Purification of the Nth Protein-- E. coli BL21 carrying pGEX-Nth was grown at 37 °C in LB medium containing ampicillin until the optical density at 600 nm reached 0.4. After the addition of 0.1 mM isopropyl beta -D-galactopyranoside (IPTG), the culture was incubated at 37 °C for 6.5 h. All subsequent procedures were carried out at 4 °C. The cells were resuspended in phosphate-buffered saline containing 0.2% Triton X-100 and then sonicated on ice. After centrifugation of the cell lysate at 15,000 × g for 30 min at 4 °C, the supernatant was applied to a glutathione-Sepharose 4B column. After washing with 50 mM Tris-HCl (pH 9.6), the GST-Nth fusion protein was eluted with 15 mM glutathione in 50 mM Tris-HCl (pH 9.6) and dialyzed against buffer A (20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 6.7 mM 2-mercaptoethanol, 0.02% Triton X-100, 300 mM NaCl). The GST-Nth fusion protein was then cleaved by thrombin by incubation for 20 h at 4 °C, and the Nth protein was purified using a spin column to remove the GST tag. The purified Nth protein was stored at -80 °C.

Proteins were analyzed by SDS-polyacrylamide gel eletrophoresis (PAGE). The concentration of protein was determined using a BCA Protein Assay Reagent Kit (Pierce) with bovine serum albumin as the standard.

Expression and Purification of the MutM Protein-- E. coli KSR7 (nth nei mutM) carrying pKK-MutM was grown at 37 °C in LB medium containing appropriate antibiotics until the optical density at 600 nm reached 0.3. After IPTG was added at a final concentration of 0.2 mM, the culture was incubated at 37 °C for 12 h. All subsequent procedures were carried out at 4 °C, unless otherwise stated. The cells were harvested and resuspended in buffer B (25 mM Tris-HCl (pH 7.6), 1 mM EDTA, 5 mM dithiothreitol, 0.25 M sucrose, 1 mM PMSF) supplemented with 1 mg/ml of lysozyme. The cells were incubated at 37 °C for 15 min and then disrupted by sonication on ice after freezing and thawing (5 cycles). The lysate was centrifuged at 11,000 × g for 60 min and subsequently at 21,000 × g for 15 min at 4 °C. After fractional precipitation of the supernatant with 65% saturation of ammonium sulfate, the precipitate was dissolved and dialyzed in buffer C (25 mM Tris-HCl (pH 7.6), 1 mM EDTA, 13 mM 2-mercaptoethanol, 0.5 mM PMSF, 5% glycerol) containing 150 mM NaCl. The dialysate was clarified by centrifugation at 11,000 × g for 30 min and loaded on a HiTrap Q Anion Exchange column. The active fractions eluted with 150-200 mM NaCl were pooled, concentrated by ammonium sulfate precipitation (75% saturation), and dialyzed against buffer D (20 mM Hepes-KOH (pH 7.6), 1 mM EDTA, 6.7 mM 2-mercaptoethanol, 0.5 mM PMSF, 5% glycerol) containing 100 mM NaCl. The dialysate was applied to a HiTrap SP Cation Exchange column, which had been equilibrated with buffer D. Proteins were eluted with a linear gradient of NaCl (100-1000 mM). The active fractions eluted between 550-650 mM NaCl were pooled and concentrated. After dialysis against buffer D containing 100 mM NaCl, the fraction was loaded onto a HiTrap Heparin column. The active fractions eluted between 600 and 700 mM NaCl were concentrated and dialyzed against buffer D containing 300 mM NaCl. The dialysate was applied to a HiLoad Superdex 75 Gel Filtration column. The active fractions eluted with buffer D containing 300 mM NaCl were subsequently applied to a Mono S column. The active fractions eluted between 800 and 900 mM NaCl were pooled and stored at -80 °C.

Expression and Purification of the Nei Protein-- Crude extracts were prepared from E. coli KSR7/pKK-Nei as described above. All procedures were carried out at 4 °C. After fractional precipitation of the supernatant with 20-65% saturation of ammonium sulfate, the precipitate was dissolved and dialyzed against buffer D containing 200 mM NaCl. The dialysate was applied to a HiTrap Q column equilibrated with buffer D containing 200 mM NaCl. The flow-through fractions were concentrated using Amicon filtration and dialyzed against buffer D containing 100 mM NaCl. The dialysate was applied to a HiTrap SP column equilibrated with buffer D containing 100 mM NaCl. Proteins were eluted with a linear gradient of NaCl (100-1000 mM). The active fractions eluted between 400 and 550 mM NaCl were pooled and dialyzed against buffer D containing 100 mM NaCl. The dialysate was applied to a HiTrap Heparin column and eluted with a linear gradient of 100-1000 mM NaCl. The active fractions eluted between 600 and 800 mM NaCl were dialyzed against buffer D containing 200 mM NaCl and applied to a HiLoad Superdex 75 column. The active fractions were then applied to a Mono S column. The active fractions were eluted between 400 and 600 mM NaCl. Purified Nei protein was stored at -80 °C

Synthesis of 5-FoU-containing Oligonucleotide Substrates-- Oligonucleotides containing a 5-foU at defined sites were synthesized as described previously (41). In brief, oligonucleotides (17- and 22-mer) containing 5-(1,2-dihydroxyethyl)uracil were synthesized by the phosphoramidite chemistry using an ABI 381A DNA synthesizer (PerkinElmer Life Sciences). Saturated NaIO4 was added to a solution of the oligonucleotide containing the precursor of 5-foU, and the reaction mixture was vortex-mixed for 1 min at room temperature to convert 5-(1,2-dihydroxyethyl)uracil to 5-foU (41). The oligonucleotides containing 5-foU were verified by HPLC and HPLC/mass spectrometry analyses. The nucleotide sequences are shown in Fig. 1.


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  		Fig. 1.   Nucleotide sequences of oligonucleotides. F represents 5-formyluracil.

Trapping Assay of Protein-Substrate Intermediates with NaBH4-- DNA trapping assays were performed at 37 °C for 30 min in a reaction mixture (10 µl) containing 10~40 fmol of 32P-labeled duplex oligonucleotides (Oligo 1/Oligo 4, Oligo 3/Oligo 4, Oligo 5/Oligo 7, or Oligo 6/Oligo 7) with crude extracts or purified Nth, Nei, or MutM protein in 20 mM Hepes-KOH (pH 7.6), 10 mM dithiothreitol, 1.5 mM MgCl2, 5 mM KCl, 2 mM EDTA, 1% glycerol, 0.5 mg of bovine serum albumin in the presence of 100 mM NaBH4. The aqueous solution of NaBH4 (1 M) was prepared just before use. The reaction was terminated by heating at 95 °C for 5 min after the addition of an equal volume of loading buffer. Trapped complexes were analyzed by 10, 12, or 15% SDS-PAGE. The gels were dried and autoradiographed using Fuji RX film at -80 °C.

DNA Glycosylase/AP Lyase Assay for 5-FoU-containing DNA-- DNA nicking assays were carried out at 37 °C in a reaction mixture (10 µl) containing 20 fmol of 32P-labeled oligonucleotides (Oligo 1/Oligo 4 or Oligo 3/Oligo 4) and purified Nth, Nei, or MutM protein in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl. The reaction was terminated by the addition of stop solution (95% formamide, 0.1% bromphenol blue, 0.1% xylene cyanol, 20 mM EDTA). After heating at 95 °C for 5 min, the samples were cooled and then loaded onto 20% polyacrylamide gels in 90 mM Tris borate (pH 8.3) containing 7 M urea and 2 mM EDTA. After electrophoresis at 1,300 V, the gels were dried and autoradiographed using Fuji RX film at -80 °C.

Assay for Mutation of 5-FoU-containing Plasmids Replicated in E. coli Cells-- Oligo 2 and Oligo 3 were phosphorylated at the 5'-end by T4 polynucleotide kinase at 37 °C for 30 min and annealed with the phosphorylated Oligo 8 strand. Vector plasmid pSVK3 was digested with KpnI and SalI, and the large fragment was isolated by electroelution and dissolved in distilled water. The double-stranded oligonucleotide containing 5-foU/A or T/A (1.75 pmol) was ligated to the large fragment (0.35 pmol) by T4 DNA ligase in 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM 2-mercaptoethanol, 200 µM ATP at 16 °C for 24 h.

The constructed plasmid DNA was introduced into E. coli AB1157, MS23 (alkA), and KSR8 (nth nei mutM alkA) by the calcium chloride method (45). After incubation at 37 °C for 1 h, an aliquot (0.1 ml) of the culture was plated on LB agar containing 50 µg/ml ampicillin and incubated at 37 °C for 18 h. The ampicillin-resistant transformants were picked and grown in LB medium containing 50 µg/ml of ampicillin at 37 °C. After 18 h, the plasmid amplified in E. coli cells was recovered by the alkaline lysis method (45). To measure the transformation efficiency, a small portion of the recovered plasmid was introduced into E. coli cells. The recovered plasmid was treated with SalI. The plasmids in the "mutant" pool were resistant to SalI. E. coli HB101 cells were transfected with the treated plasmid by electroporation using a Gene Pulser Transfection Apparatus with a Pulse Controller (Bio-Rad). After overnight incubation at 37 °C, the number of colonies obtained with digested DNA (N) and that obtained with undigested DNA (N0) was counted to calculate the ratio A = N/N0. The mutation in the recovered progeny plasmids was confirmed by digestion with the restriction enzyme. Furthermore, the nucleotide sequence was determined with a sequencing primer and an ABI PRISM BigDye Terminator Cycle Sequencing Kit (PerkinElmer Life Sciences) using an ABI 310 Genetic Analyzer (PerkinElmer Life Sciences). The ratio B ((the number of mutant plasmids that were resistant to restriction enzyme and had mutated nucleotide sequence)/(the number of plasmids assayed)) was determined. The mutation frequency of plasmid (F) was calculated as follows: F = A × B.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Proteins with 5-FoU-DNA Glycosylase/AP Lyase Activity in Extracts from E. coli by the Borohydride Trapping Assay-- We first carried out the borohydride trapping assay to detect and characterize proteins with DNA glycosylase/AP lyase activity for 5-foU in E. coli extracts. This assay is based on the fact that DNA glycosylases/AP lyases form a Schiff base intermediate that can be trapped by NaBH4 to generate a stable covalent enzyme-DNA complex (35-40). Crude extracts prepared from E. coli were incubated with 5'-32P-labeled duplex oligonucleotide containing a single 5-foU/A or T/A in the presence of 100 mM NaBH4. Trapped DNA-protein complexes were analyzed by SDS-PAGE. Fig. 2 shows that, with the oligonucleotide containing 5-foU, some shifted bands were detected in the extracts from wild-type strain (lane 1). The formation of the complexes was independent of MutS and AlkA proteins, because these shifted bands were found in extracts from the alkA and mutS mutants (lanes 3 and 5). No shifted bands were detected with T/A oligonucleotide (lanes 2, 4, and 6). The trapped complexes disappeared upon boiling the reaction mixtures (data not shown), indicating that the shifted bands were due to 5-foU-specific proteins present in the extracts of E. coli.


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  		Fig. 2.   Formation of covalent complexes of proteins in extracts from E. coli and 5-foU-containing oligonucleotide in the presence of NaBH4. Oligonucleotide substrates containing 5-foU and thymine (Oligo 1/Oligo 4 and Oligo 3/Oligo 4) were incubated with extracts (15 µg of protein) from E. coli wild type (lanes 1 and 2), alkA (lanes 3 and 4), and mutS (lanes 5 and 6) in the presence of 100 mM NaBH4 at 37 °C for 30 min, and the products were subjected to 12% SDS-PAGE. Lanes 1, 3, and 5, 5-foU-containing oligonucleotide; lanes 2, 4, and 6, thymine-containing oligonucleotide. Shifted bands (trapped complexes) and free oligonucleotides are indicated.

Trapping Assay with Extracts from Various DNA Repair Mutants of E. coli-- Next, trapping assays were performed with extracts from various DNA repair mutants of E. coli to identify genes responsible for the formation of the trapped complexes with the 5-foU-containing oligonucleotides. The extracts were incubated with duplex oligonucleotides containing 5-foU/A (Oligo 1/Oligo 4) in the presence of NaBH4 (Fig. 3). After extensive electrophoresis on 15% polyacrylamide gels, extracts from E. coli wild-type strains showed the formation of three trapped complexes (lane 1): a high mobility complex (band 1), a middle mobility complex (band 2), and a low mobility complex (band 3). Mutation of the nth gene resulted in selective loss of band 1 (lanes 2, 6, and 7). On the other hand, mutation of the nei and mutM genes resulted in complete loss of band 2 (lanes 3, 5, and 7) and band 3 (lanes 4-6), respectively. With the extract of the nth nei mutM triple mutant, all the bands completely disappeared (lane 8).


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  		Fig. 3.   Formation of covalent complexes of proteins in extracts from E. coli nth, nei, and mutM mutants and E. coli KSR7 (nth nei mutM) strain carrying pACYCnth, pACYCnei, and pKK-MutM and 5-foU-containing oligonucleotide in the presence of NaBH4. Oligonucleotide substrate containing 5-foU (Oligo 1/Oligo 4) was incubated with extracts (15 µg of protein) from E. coli wild-type and various mutant strains in the presence of 100 mM NaBH4 at 37 °C for 30 min, and the products were subjected to 15% SDS-PAGE. Lane 1, CSR26 (wild type); lane 2, KSR1 (nth); lane 3, KSR2 (nei); lane 4, KSR3 (mutM); lane 5, KSR6 (nei mutM); lane 6, KSR5 (nth mutM); lane 7, KSR4 (nth nei); lane 8, KSR7 (nth nei mutM); lane 9, KSR7/pACYCnei; lane 10, KSR7/pACYCnth; lane 11, KSR7/pKK-MutM.

Next, plasmids pACYCnth, pACYCnei, and pKK-MutM were introduced into E. coli KSR7 (nth nei mutM), and extracts were prepared and incubated with the duplex Oligo 1/Oligo 4 in the presence of NaBH4. As shown in Fig. 3, the introduction of the nth, nei, and mutM genes into E. coli KRS7 restored the corresponding bands 1, 2, and 3, respectively (Fig. 3, lanes 9-11). These results indicated that the Nth, Nei, and MutM proteins have a DNA glycosylase/AP lyase activity that recognizes and forms a covalent complex with the 5-foU-containing oligonucleotide.

Other DNA repair mutations tested, including alkA, mutS, mutL, mutH, mutY, mutT, ada, ogt, uvrA, uvrB, uvrC, vsr, dps, nfo, xthA, recA, recB, recC, dut, ung, and uvrD, did not affect the formation of the DNA-protein complexes (data not shown). No shifted bands were detected for the T/A base pair (data not shown). These results established that bands 1, 2, and 3 consisted of the MutM, Nei, and Nth proteins, respectively. Because similar results were obtained with the duplex oligonucleotide (Oligo 5/Oligo 7) (data not shown), we used Oligo 1/Oligo 4 for further experiments.

Trapping Activity of the Purified Nth, Nei, and MutM Proteins to the 5-FoU DNA-- Next, we purified Nth, Nei, and MutM proteins from E. coli cells to examine whether these enzymes are directly trapped by 5-foU-containing DNA and whether they can cleave the 5-foU-containing oligonucleotide at the 5-foU site. The entire open reading frame of the nth gene was amplified by PCR and subcloned into pGEX-4T-1 to obtain the GST-Nth fusion protein. The fusion protein was expressed in E. coli BL21/pGEX-Nth with IPTG and purified by means of glutathione-Sepharose 4B column chromatography. Purified Nth protein gave a single band with an apparent molecular mass of 23.6 kDa in a SDS-polyacrylamide gel (Fig. 4A). The purified Nth protein was used for further experiments.


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  		Fig. 4.   Expression and purification of the Nth, Nei, and MutM proteins. A, E. coli BL21 carrying pGEX-Nth was induced by 0.1 mM IPTG for 6.5 h. Proteins were separated by 15% SDS-PAGE and stained with Coomassie Blue. Lane M, molecular weight marker proteins; lane 1, soluble fraction disrupted by sonication; lane 2, flow-through fraction from glutathione-Sepharose column; lane 3, purified GST-Nth fusion protein after elution from GST-Sepharose with 15 mM glutathione; lane 4, purified Nth protein after digestion with thrombin and elution from glutathione Sepharose 4B MicroSpin column. B, E. coli KSR7 (nth nei mutM) carrying pKK-MutM was induced with 0.2 mM IPTG for 12 h. Proteins were separated by 15% SDS-PAGE and stained with Coomassie Blue. Lane M, protein marker; lane 1, soluble fraction disrupted by sonication; lane 2, 65% ammonium sulfate saturation fraction; lane 3, active fraction after HiTrap Q anion exchange column chromatography; lane 4, active fraction after HiTrap SP cation exchange column chromatography; lane 5, active fraction after HiTrap Heparin column chromatography; lane 6, active fraction after HiLoad Superdex 75 pg gel filtration column chromatography; lane 7, active fraction after Mono S column chromatography. C, E. coli KSR7 (nth nei mutM) carrying pKK-Nei was induced with 0.2 mM IPTG for 12 h. Proteins were separated by 15% SDS-PAGE and stained with Coomassie Blue. Lane M, protein marker; lane 1, soluble fraction disrupted by sonication; lane 2, 20-65% ammonium sulfate saturation fraction; lane 3, active fraction after HiTrap Q anion exchange column chromatography; lane 4, active fraction after HiTrap SP Cation Exchange column chromatography; lane 5, active fraction after HiTrap Heparin column chromatography; lane 6, active fraction after HiLoad Superdex 75 pg gel filtration column chromatography; lane 7, active fraction after MonoS column chromatography.

The results of trapping assays are shown in Fig. 5 (A and B, lanes 7-12). Fig. 5A shows that the purified Nth protein was trapped to the oligonucleotide containing 5- foU/A by NaBH4 (lane 6) but not to the T/A oligonucleotide (lane 2). These results indicated that the Nth protein could form the trapped complex with the 5-foU-containing DNA.


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  		Fig. 5.   Borohydride trapping analysis of purified Nth, Nei, and MutM proteins. A, oligonucleotide substrates containing 5-foU and thymine (Oligo 1/Oligo 4 and Oligo 3/Oligo 4) (20 fmol) were incubated with purified Nth, Nei, or MutM protein (20, 6.6, or 128 pmol) in the presence of 100 mM NaBH4 at 37 °C for 30 min. Shifted bands (trapped complexes) and free oligonucleotides are indicated. Lanes 1-4, thymine-containing oligonucleotide; lanes 5-8, 5-foU-containing oligonucleotide. Lanes 1 and 5, no protein; lanes 2 and 6, Nth protein; lanes 3 and 7, Nei protein; lanes 4 and 8, MutM protein. B, oligonucleotide substrate containing 5-foU (Oligo 1/Oligo 4) was incubated with various concentrations of purified Nth, Nei, or MutM protein in the presence of 100 mM NaBH4 at 37 °C for 30 min, followed by SDS-PAGE analysis. Shifted bands (trapped complexes) and free oligonucleotides are indicated. Lanes 1-6, MutM (lane 1, 0 pmol; lane 2, 3.2 pmol; lane 3, 6.4 pmol; lane 4, 32 pmol; lane 5, 64 pmol; lane 6, 128 pmol); lanes 7-12, Nth (lane 7, 0 pmol; lane 8, 0.5 pmol; lane 9, 1 pmol; lane 10, 5 pmol; lane 11, 10 pmol; lane 12, 20 pmol); lanes 13-18, Nei (lane 13, 0 pmol; lane 14, 0.33 pmol; lane 15, 1.65 pmol; lane 16, 3.3 pmol; lane 17, 9.9 pmol; lane 18, 13.2 pmol).

Next, the MutM protein was purified from extracts of E. coli KSR7 (nth nei mutM)/pKK-MutM by ammonium sulfate precipitation, followed by chromatography through HiTrap Q, HiTrap SP, HiTrap Heparin, HiLoad Superdex 75, and Mono S. MutM protein showed homogeneous molecular mass of ~31 kDa in SDS-PAGE (Fig. 4B). Fractions were monitored for 5-foU DNA glycosylase/AP lyase activity by NaBH4, and proteins were analyzed by SDS-PAGE. The ability of MutM protein to cleave an 8-oxoguanine-containing duplex oligonucleotide was also assayed (data not shown). It was evident that the MutM protein also had the ability to form a trapped complex with the 5-foU-containing oligonucleotide (Fig. 5, A, lanes 4 and 8, and B, lanes 1-6).

The Nei protein was also purified from E. coli KSR7 (nth nei mutM)/pKK-Nei as described above. Each purification step was monitored for 5-foU DNA glycosylase/AP lyase activity by NaBH4, and proteins were analyzed by SDS-PAGE. The Nei protein showed a homogeneous molecular mass of ~29 kDa in SDS-PAGE (Fig. 4C). The Nei protein also had an activity to form the trapped complex with the 5-foU-containing oligonucleotide by NaBH4 (Fig. 5, A, lanes 3 and 7, and B, lanes 13-18).

The formation of trapped complexes increased with the amount of the purified Nth, Nei, and MutM proteins (Fig. 5B). No protein-DNA complex was formed when heat-denatured Nth, Nei, and MutM proteins were incubated with the 5-foU-containing oligonucleotide in the presence of NaBH4 (data not shown).

Cleavage of 5-FoU-containing Oligonucleotide by the Purified Nth, Nei, and MutM Proteins-- The Nth, Nei, and MutM proteins are DNA glycosylases/AP lyases that catalyze both the cleavage of the glycosylic bond to release damaged bases and the incision of the phosphodiester backbone at the resulting AP site via beta  or beta - and delta -elimination reactions (5, 12, 20-23). To make it more evident that the Nth, Nei, and MutM proteins indeed have a DNA glycosylase/AP lyase activity resulting in release of the 5-foU from DNA, a DNA nicking assay was performed. We first analyzed the products of the cleavage reaction with the proteins. Duplex oligonucleotide (Oligo 1/Oligo 4) containing 5-foU/A was incubated with the proteins at 37 °C, followed by analysis of the products by polyacrylamide gel electrophoresis. The results are shown in Fig. 6A. The Nth protein was found to cleave the oligonucleotide at the site of 5-foU by a beta -elimination reaction (lane 2), whereas the Nei and MutM proteins by beta - and delta -elimination reactions (lanes 3 and 4), by comparing the mobility on gels with the marker oligonucleotides (Oligo 9 and Oligo 10) (data not shown). The oligonucleotide with T/A was not cleaved by the proteins (data not shown). Sometimes the beta -elimination products generated by AP lyases, e.g. endonuclease III, give rise to several separate bands in PAGE analysis, presumably because of Tris-adduct formation (23, 46) and/or isomerization of the 3'-hydroxypentenal terminus (21, 47). These results indicate that 5-foU is recognized and removed by the enzymes as a natural substrate.


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  		Fig. 6.   Cleavage of 17-mer oligonucleotide containing 5-foU by the Nth, Nei, and MutM proteins. A, the 5-foU-containing duplex oligonucleotide (Oligo 1/Oligo 4) (20 fmol) was incubated at 37 °C for 15 min without protein (lane 1), or with Nth (5 pmol, lane 2), Nei (1.65 pmol, lane 3), or MutM (32 pmol, lane 4). The products were separated by denaturing 20% PAGE in gels containing urea. B, the 5-foU-containing duplex oligonucleotide (Oligo 1/Oligo 4) (20 fmol) was incubated at 37 °C for 5 min without protein (lanes 1, 6, and 11), or with MutM (lanes 2-5), Nei (lanes 7-10), or Nth (lanes 12-15). The proteins were diluted just before use. The products were separated by denaturing 20% PAGE in gels containing urea. Lanes 2-5, 3.2, 6.4, 12.8 and 32 pmol, respectively; lanes 7-10, 0.165, 0.33, 0.66 and 1.65 pmol, respectively; lanes 12-15, 0.5, 1, 2 and 5 pmol, respectively.

Second, the duplex oligonucleotide containing 5-foU/A was incubated with the proteins for various times up to 60 min. The substrate oligonucleotide was almost completely cleaved by the Nth, Nei, and MutM proteins when incubated for 5 min. Next, the cleavage reaction was carried out at 37 °C for 5 min with the proteins. As seen in Fig. 6B, the 17-mer oligonucleotide was cleaved by all tested amounts of the proteins. Complete cleavage of the oligonucleotide occurred with 1, 0.66, and 6.4 pmol of the Nth, Nei, and MutM proteins, respectively. The specific activities of the Nth, Nei, and MutM proteins were 0.6, 0.91, and 0.094 nmol/pmol protein/h, respectively. From these results, it is likely that the Nei protein most efficiently cleaves the 5-foU-containing oligonucleotide.

Mutation Frequency of the Plasmid Containing 5-FoU in E. coli AB1157, MS23, and KSR8 Strains-- Biochemical studies with purified Nth, Nei, and MutM proteins revealed that these proteins have a DNA glycosylase/AP lyase activity that acts on 5-foU in DNA. To clarify the role of these proteins in preventing mutations because of 5-foU in vivo, we determined the frequencies of mutations of 5-foU-containing plasmid pSVK3 that occurred during replication in E. coli with various DNA repair capacities. We introduced 5-foU into a unique SalI site (Fig. 1, Oligo 2/Oligo 8). Mutations were assayed as follows: plasmids replicated in E. coli were digested by SalI. Plasmid DNA in linear form cannot be replicated in E. coli HB101, which carries the recA mutation. Therefore, the HB101 cells that are resistant to ampicillin must carry mutant plasmids that were resistant to SalI. When the plasmid containing 5-foU was replicated in the nth nei mutM alkA mutant, the mutation frequency was significantly increased (~10 fold), compared with that in wild-type and alkA strains (Table I). These results indicated that 5-foU is one of the substrates of the Nth, Nei, and MutM proteins and that the proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli cells.

                              
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Table I
Mutation frequencies of plasmid containing 5-foU in E. coli AB1157, MS23 (alkA), and KSR8 (nth nei mutM alkA)
Each value represents the mean of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 5-foU is suggested to be a potentially mutagenic lesion (26-30). 5-Formyl-2'-deoxyuridine and 5-formyl-deoxyUTP exogeneously added to the culture medium cause the mutagenic effects in bacterial cells (26, 27). Moreover, in this study, we showed a mutagenic effect when the 5-foU-containing plasmid was replicated in E. coli cells. These results showed conclusively that 5-foU is a mutagenic lesion. These observations led us to postulate that E. coli cells must have repair mechanisms for the oxidative lesion to avoid mutations.

In this study, using the borohydride trapping assay, we identified proteins in E. coli extracts that act on 5-foU in DNA. This assay is based on the fact that all known DNA glycosylases/AP lyases, irrespective of their structure, use an enzyme-derived amine nucleophile to expel the aberrant base (5, 12, 35-40). This results in a Schiff base intermediate, which can be trapped by borohydride, that results in irreversible DNA-protein cross-linking (35-40). Detection of the trapped protein-DNA complex is useful to identify not only DNA glycosylases with AP lyase activity but also to delineate the functional domains (35-40). Therefore, we used this assay to identify enzymatic activities that recognize 5-foU in extracts from E. coli cells. We found that Nth, Nei, and MutM proteins had novel DNA glycosylase/AP lyase activities that recognize and remove 5-foU in DNA. This conclusion was derived from the following facts: these proteins could be trapped by NaBH4 to the 5-foU-containing oligonucleotide (Figs. 3 and 5) and efficiently cleaved the oligonucleotide at the 5-foU site (Fig. 6). The purified Nth protein excision of the 5-foU-containing oligonucleotide showed efficiency similar to that observed with thymine glycols (48). In addition, the facts that the Nth protein cleaved the oligonucleotide by a beta -elimination reaction indicated that the enzyme acts on 5-foU through the same mechanism as that for substrates previously characterized such as thymine glycols and urea. The same argument might apply for the functions of the Nei and MutM proteins. However, the trapping assay is not suited for assaying DNA glycosylase without AP lyase activity, like E. coli AlkA. Hence, it is not excluded that E. coli has another repair enzyme(s) operating on 5-foU.

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death (5, 6, 11-13). Nth protein (endonuclease III) was first identified as an activity in E. coli that introduced strand incision in X-irradiated, UV-irradiated and OsO4-treated DNA (5, 6, 49, 50). This enzyme was characterized as a DNA glycosylase specific for damaged thymine such as thymine glycols and urea. Recently, a wide variety of additional substrates have been identified for the Nth protein. These Nth substrates share the common feature of having resulted from oxidative damage to pyrimidines. They include urea, thymine glycols, methyl tartonylurea, 5-hydroxy-5-methylhydatoin, 5,6-dihydrothymine, 5-hydro-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5,6-dihydrouracil, uracil glycol, 6-hydroxy-5,6-dihydrocytidine, 5-hydroxycytine, and 5-hydroxyuracil (6, 51, 52). The relative activity of Nth protein toward many of these substrates and their in vivo importance remain to be clarified. In this study, we demonstrated a novel activity of the Nth protein that recognizes and repairs 5-foU using a defined oligonucleotide substrate (28, 29, 41). A common feature of the identified Nth substrates is that they are no longer aromatic and planar in the pyrimidine ring (5, 6, 12). Exceptions to this include 5-hydroxycytosine and 5-hydrouracil (5, 6, 12) and 5-foU. The active site of Nth protein must be able to accommodate this wide variety of substrates, including 5-foU (6). It will be of interest to compare the structure of the active site pocket of the Nth protein (12) when bound to thymine glycol and 5-foU.

MutM protein is an E. coli enzyme responsible for the removal of formamidepyrimidine and 8-hydroxyguanine in DNA (5, 6, 12, 53, 54). A variety of other substrates have been identified for the MutM protein. It recognizes and removes 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2,6-diamono-4-hydroxy-5-N-methylformamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine (5, 6, 55, 56). However, the importance of these substrates in vivo is still unknown. These substrates provide insight into the mechanism of damage recognition by the MutM protein. Recently, 5-hydroxycytosine and 5-hydroxyuracil have been found to be released by the MutM protein (5, 55, 56). Furthermore, in this study, we found that 5-foU is also a substrate of the MutM protein. It is possible that the MutM protein is able to accommodate these small oxidized pyrimidines within its active site because it might be designed for larger oxidized purines (6).

The Nei protein showed significant homology with MutM protein, especially in the N- and C-terminal regions (5, 6, 16, 19). The two proteins also have similar sizes, similar molecular masses, and similar hydrophobicities (6, 12, 19). Nei protein also shares several catalytic activities with MutM protein, including cleavage of DNA by beta , delta -elimination (5, 6, 11-13, 20-23). Nei protein recognizes premutagenic pyrimidine lesions such as 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol (16). Recently, Blaisdell et al. (58) and Hazra et al. (59) have shown that the Nei protein (endonuclease VIII) has 8-oxoguanine-DNA glycosylase activity. In addition, in this study, we found that 5-foU is a good substrate for the Nei protein (Figs. 5 and 6). The purified Nei protein showed a high efficiency of removing 5-foU from duplex DNA. The fact that the Nth, Nei, and MutM proteins recognize many types of base modifications, irrespective of their structure, gives insight into the common mechanism of recognition of the substrate by these enzymes.

In this study, we found that the frequency of mutations of the 5-foU-containing plasmid significantly increased when it was replicated in E. coli KSR8 (nth nei mutM alkA), compared with in wild-type and alkA strains (Table I). Single mutations in the nth, nei, or mutM gene did not affect the mutation frequency, like the alkA mutation (data not shown). Thus, these gene products might be back-up enzymes to repair 5-foU in DNA.

Base excision repair is highly conserved from bacteria to humans (5, 6, 12, 60). Human hNTH1 and mouse mNTH proteins are structural and functional homologues of E. coli endonuclease III (57, 61, 62). mNTH does not act on 5-foU-containing oligonucleotides (14). On the other hand, in this study, hNTH1 protein was found to have trapping activity to the 5-foU oligonucleotide (data not shown).

    Ackowledgements

We thank Drs. K. Yamamoto (Tohoku University, Sendai, Japan), B. Weiss (Michigan University, Ann Arbor, MI), A. Nishimura (National Institute of Genetics, Mishima, Japan), R. P. Cunningham (State University of New York, Albany, NY), M. M. Wu (Harvard University, Cambridge, MA), A. Holmgren (Karolinska Institute, Stockholm, Sweden), B. Bachmann (Yale University, New Haven, CT), D. Touati (Paris University, Paris, France) and H. Shinagawa (Osaka University, Osaka, Japan) for kindly supplying E. coli strains and plasmids.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan and from the Nissan Science Foundation (to Q.-M. Z.). This work was also supported by a grant from the Core Research for Evolutionary Science and Technology of the Japan Science and Technology Corporation (to H. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-4097; Fax.: 81-75-753-4087; E-mail: yonei@kingyo.zool.kyoto-u.ac.jp.

Published, JBC Papers in Press, August 23, 2000, DOI 10.1074/jbc.M006125200

    ABBREVIATIONS

The abbreviations used are: AP, apurinic/apyrimidinic; 5-foU, 5-formyluracil; IPTG, isopropyl-beta -D-galactopyranoside; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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M. Hori, S. Yonei, H. Sugiyama, K. Kino, K. Yamamoto, and Q.-M. Zhang
Identification of high excision capacity for 5-hydroxymethyluracil mispaired with guanine in DNA of Escherichia coli MutM, Nei and Nth DNA glycosylases
Nucleic Acids Res., February 15, 2003; 31(4): 1191 - 1196.
[Abstract] [Full Text] [PDF]

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M. Takao, S.-i. Kanno, K. Kobayashi, Q.-M. Zhang, S. Yonei, G. T. J. van der Horst, and A. Yasui
A Back-up Glycosylase in Nth1 Knock-out Mice Is a Functional Nei (Endonuclease VIII) Homologue
J. Biol. Chem., October 25, 2002; 277(44): 42205 - 42213.
[Abstract] [Full Text] [PDF]

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 Nucleic Acids ResHome page
I. Miyabe, Q.-M. Zhang, K. Kino, H. Sugiyama, M. Takao, A. Yasui, and S. Yonei
Identification of 5-formyluracil DNA glycosylase activity of human hNTH1 protein
Nucleic Acids Res., August 1, 2002; 30(15): 3443 - 3448.
[Abstract] [Full Text] [PDF]

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