Fidelity of Uracil-initiated Base Excision DNA Repair in Escherichia coli Cell Extracts*

Jung-Suk SungDagger , Samuel E. BennettDagger , and Dale W. MosbaughDagger §||

From the Departments of Dagger  Environmental and Molecular Toxicology and § Biochemistry and Biophysics and the  Environmental Health Science Center, Oregon State University, Corvallis, Oregon 97331-7301

Received for publication, September 6, 2000, and in revised form, October 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung+ dug+), ~80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung- dug+) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung- dug+), BH157 (ung+ dug-), and BH158 (ung- dug-) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be ~5.5 × 10-4 with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Uracil-mediated base excision DNA repair serves as a strategic cellular defense mechanism to maintain the genetic stability of the Escherichia coli genome (1). This BER1 process protects the DNA from premutagenic U·G2 mispairs formed by cytosine deamination and U·A base pairs produced by incorporation of dUMP during DNA synthesis (2, 3). BER is initiated when uracil-DNA glycosylase recognizes a uracil residue in DNA and catalyzes the cleavage of the N-glycosylic bond that links the uracil base to the deoxyribose phosphate DNA backbone (4). This hydrolytic reaction results in the release of free uracil and creates an abasic site in the DNA (4). Incision by a class II AP endonuclease, either endonuclease IV (Nfo) (5) or exonuclease III (Xth) (6), cleaves the phosphodiester bond on the 5'-side of the AP site to generate a terminal 3'-hydroxyl-containing nucleotide and a deoxyribose 5'-phosphate residue (7). Approximately 90% of the AP endonuclease activity detected in E. coli is accounted for by the Xth protein (8). Removal of the dRP moiety prior to gap-filling DNA synthesis can occur by the deoxyribophosphodiesterase activity of RecJ, which also possesses a 5' to 3' single-stranded DNA exonuclease (9, 10). In addition, the product of the mutM gene (Fpg) has been reported to catalyze the 5' to 3' removal of incised dRP residues by a beta -elimination mechanism (11). Furthermore, exonuclease I (SbcB) has been shown to release dRP from an AP site incised by Nfo and also to remove 4-hydroxy-2-pentenal-5-phosphate from an abasic site incised by AP lyase on the 3'-side of the lesion (12). Collectively, RecJ, Fpg, and SbcB may be responsible for dRP removal in E. coli, since inactivation of all three dRP excision activities produced a lethal phenotype (12). In association with the dRP excision step, one or more nucleotides may be removed from the uracil-containing DNA strand by a 5' to 3' exonuclease activity. However, appreciable degradation of the incision site in the 3' to 5' direction does not seem to occur (13). Gap-filling DNA synthesis replaces the excised nucleotide(s) by the action of a DNA polymerase, and DNA ligase re-establishes the continuity of the phosphodiester backbone (10).

In E. coli, two genetically distinct forms of uracil-DNA glycosylase have been purified to apparent homogeneity, characterized, and demonstrated to initiate uracil-mediated BER (4, 14-17). E. coli uracil-DNA glycosylase (Ung) was the first DNA glycosylase identified and consists of a monofunctional single polypeptide with a molecular mass of 25,558 daltons (18, 19). Ung prefers to act on uracil residues located in single-stranded DNA but also recognizes uracil in duplex DNA with moderately reduced efficiency (20). A second protein, referred to as double-stranded uracil-DNA glycosylase (Dug, also termed Mug), was recently purified as a 18,672 molecular weight polypeptide and characterized (14, 16). Based on amino acid sequence alignment, Dug lacks strong sequence identity (~10%) with Ung but shares remarkable similarity in tertiary structure (17). Dug preferentially removes uracil from DNA containing a U·G or U·T mispair but inefficiently recognizes a U·A base pair target and lacks detectable activity on single-stranded uracil-containing DNA (14, 16).

The activity of Ung can also be differentiated from that of Dug based on several other biochemical properties: (i) Ung activity is inhibited by the PBS-1 and -2 uracil-DNA glycosylase inhibitor (Ugi) protein, which forms an essentially irreversible Ung·Ugi complex (21), whereas Dug activity is insensitive to Ugi (14, 17, 22); (ii) purified Dug is a relatively inefficient enzyme that exhibits a significantly lower uracil excision turnover number than Ung (4, 14, 17); (iii) Dug is strongly inhibited by apyrimidinic sites but not by free uracil, whereas Ung exhibits modest inhibition by both reaction end products (14); and (iv) Dug efficiently cleaves 3,N4-ethenocytosine from duplex DNA, while this exocyclic DNA adduct remains refractory to removal by Ung (14, 16). The involvement of Ung in uracil-DNA repair has been well documented (1, 13, 23); however, the role of Dug remains to be fully elucidated. Sung and Mosbaugh (14) recently demonstrated the involvement of Dug in BER using an E. coli NR8052 (ung) cell-free extract and an M13mp2 form I DNA substrate containing a site-specific U·G mispair. Uracil-initiated BER conducted in the absence of Ung was shown to be both insensitive to Ugi and stimulated by the addition of exogenous Dug (14). Thus, while it has been established that E. coli possesses two classes of uracil-DNA glycosylase capable of initiating BER, the relative contribution of Ung and Dug in mediating repair remains to be determined.

Studies of the repair patch size associated with gap-filling DNA synthesis in conjunction with uracil-initiated BER have been conducted using both E. coli cell-free extracts and reconstituted enzyme systems (13, 23, 24). Two types of repair patch size have been described involving either the incorporation of a single nucleotide (short patch) or 2-19 nucleotides (long patch). Dianov et al. (13) reported that BER initiated at a U·G target site contained in an oligonucleotide (30-mer) DNA substrate was mainly repaired by a short patch process in wild-type E. coli NH5033 cell extracts. Similar results were obtained using the same DNA substrate and a reconstituted enzyme system composed of Ung, Nfo, RecJ, polymerase I, and ligase (24). In these experiments, short patch BER was largely dependent on the presence of RecJ (24). In sharp contrast, Sandigursky et al. (23) reported that E. coli AB1157 cell extracts did not efficiently support short patch repair when a closed circular DNA containing a U·G mispair was used as substrate. In this case, long patch BER was observed that was not dependent on either SbcB and RecJ or Fpg and RecJ (23). Given the apparent disparity between these two reports, further investigation into the BER patch size is warranted.

The biochemical steps involved in E. coli uracil-initiated BER have been elucidated in substantial detail. However, an assessment of the fidelity of DNA repair synthesis associated with the completed BER process initiated by either E. coli Ung or Dug has not been made. In the present study, we have (i) used a site-specific uracil-containing circular duplex DNA substrate to monitor the relative rate of Ung- and Dug-initiated BER in E. coli cell extracts; (ii) determined the repair patch size associated with BER initiated by the two uracil-DNA glycosylases; (iii) measured the base substitution error frequency produced during BER; and (iv) assessed the error specificity associated with Ung- and Dug-mediated BER.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- PCR primers, GTGTGGAATTGTGAGCGG (FP-18-mer) and CGTGCATCTGCCAGTTTG (RP-18-mer), and sequencing primer, GCACTCCAGCCAGCTTTCCGG (S-21-mer), were obtained from Research Genetics, Inc. Two oligodeoxynucleotides, CCCAGTCACGTCUTTGTAAAACG (U-23-mer) and CCCAGTCACGTCATTGTAAAACG (A-23-mer) were synthesized and purified by Oligos Etc; 5'-end-phosphorylated oligodeoxynucleotides were prepared as described previously (25).

E. coli strains NR8051 and NR8052, NR9162, and CSH50 were provided by T. A. Kunkel (NIEHS, National Institutes of Health), and E. coli BH156, BH157, and BH158 were obtained from A. S. Bhagwat (Wayne State University). E. coli JM109 was procured from New England Biolabs. P1 lysate containing mutS::Tn-10 was obtained from J. Hays (Oregon State University), and E. coli mutS strains, NR80511 and NR80521 were constructed by the P1 transduction of mutS::Tn-10 into E. coli NR8051 and NR8052, respectively.

E. coli uracil-DNA glycosylase (fraction V) and Ugi (fraction IV) were purified as described by Sanderson and Mosbaugh (26). Double-stranded uracil-DNA glycosylase (fraction VI) was isolated as described by Sung and Mosbaugh (14). E. coli endonuclease IV (fraction V) was provided by B. Demple (Harvard University), and T4 DNA polymerase was purified as described previously (25). T4 polynucleotide kinase, restriction endonuclease HinfI, and T4 DNA ligase were purchased from New England Biolabs. Proteinase K and creatine phosphokinase (type I, rabbit) were from Sigma, and ribonuclease A was from Worthington.

Preparation of Base Excision Repair DNA Substrates-- M13mp2op14 DNA that contained an opal codon located at nucleotide positions 78-80 of the lacZalpha gene was constructed and isolated as described previously by Sanderson and Mosbaugh (25), with minor modifications. Briefly, M13mp2op14 phage were propagated in E. coli JM109 cells, and single-stranded M13mp2op14 DNA was purified using the CTAB DNA precipitation method (27). Single-stranded M13mp2op14 DNA was then annealed to 5'-end-phosphorylated oligodeoxynucleotide U-23-mer or A-23-mer, and the primed template DNA was subjected to a primer extension reaction. Primer extension reaction mixtures (3030 µl) contained 20 mM Hepes-KOH (pH 7.4), 2 mM dithiothreitol, 10 mM MgCl2, 1 mM ATP, a 500 µM concentration each of dATP, dTTP, dGTP, and dCTP, 400 units of T4 DNA polymerase, 40,000 units of T4 DNA ligase, and 200 pmol of primed template DNA containing either U·T mispair or A·T base pair were incubated for 4 h at 37 °C. Covalently closed circular duplex DNA reaction products were isolated by ethidium bromide cesium chloride gradient centrifugation, as described previously (25). Form I DNA was isolated, extracted four times with an equal volume of 1-butanol saturated with 5 M NaCl, concentrated using a Centricon-30 concentrator (Amicon), and buffer-exchanged into TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The isolated DNA3 was >95% form I as determined by 0.8% agarose gel electrophoresis.

Preparation of E. coli Cell-free Extracts-- E. coli cells grown at 37 °C in 500 ml of YT medium (0.5% yeast extract, 0.8% tryptone, and 0.5% NaCl) to midlog phase were harvested by centrifugation at 7000 × g for 15 min at 4 °C. The cell pellet was resuspended in 10 ml of sonification buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.1 mM dithiothreitol, and cells were lysed on ice by sonification. After cell debris was removed by centrifugation at 20,000 × g for 20 min at 4 °C, protein was precipitated from the supernatant by the addition of 0.35 g of powdered ammonium sulfate per ml of extract, and the precipitate was recovered by centrifugation at 20,000 × g for 20 min at 4 °C. The pellet was resuspended in 5 ml of R-buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10% (w/v) glycerol and dialyzed against the same buffer, and the protein concentration of the cell free extract was determined by the Bradford reaction (28) using the Bio-Rad protein assay.

Base Excision DNA Repair Reaction-- Standard BER reaction mixtures (100 µl) containing 100 mM Tris-HCl (pH 7.5); 5 mM MgCl2; 1 mM dithiothreitol; 0.1 mM EDTA; 2 mM ATP; 0.5 mM beta -NAD; a 20 µM concentration each of dATP, dTTP, dGTP, and dCTP; 5 mM phosphocreatine di-Tris salt; 200 units/ml phosphocreatine kinase; 10 µg/ml M13mp2op14 (U·T) heteroduplex or (A·T) homoduplex DNA (form I); and 1 mg/ml of E. coli cell-free extract protein were prepared on ice. In some experiments, 400 µCi/ml [alpha -32P]dATP (6000 Ci/mmol) was also included in the reaction mixture. Following incubation at 30 °C in the presence or absence of 1000 units of Ugi, the reactions were terminated after various times by adjustment to 20 mM EDTA and heated at 70 °C for 3 min. RNase A was then added to 80 µg/ml, and the reaction mixtures were incubated at 37 °C for 10 min. Each reaction was then adjusted to 0.5% SDS, proteinase K was added to 190 µg/ml, and each reaction was incubated for 30 min at 37 °C. The M13mp2op14 DNA was then isolated and resuspended in 20 µl of TE buffer as described previously (25).

Analysis of Base Excision DNA Repair Reaction Product-- DNA samples (5 µl), isolated as described above, were removed and treated with 100 units of Ung for 30 min at 37 °C. After quenching the reaction by adding 1000 units of Ugi, samples were further incubated with 1 unit of Nfo for 30 min at 37 °C. Nfo was inactivated by heating at 70 °C for 3 min, and form I DNA that was resistant to Ung/Nfo cleavage was resolved from form II DNA by 0.8% agarose gel electrophoresis (25). The amount of form I and II DNA was determined by comparing the fluorescence at 300 nm of the ethidium bromide-stained DNA reaction products with that of coelectrophoresced form I and II DNA (6.25-100-ng) standards. The fluorescence intensity data were captured using a gel documentation system (Ultra Violet Products Ltd.) and quantified with the ImageQuant computer program (Molecular Dynamics, Inc., Sunnyvale, CA). After correcting for ~0.7-fold reduced staining intensity of form I DNA relative to form II DNA, the percentage of form I DNA was calculated by dividing the amount of form I (ng) by that of form I plus form II DNA and multiplying by 100.

Analysis of Base Excision Repair DNA Synthesis-- Standard BER reactions were conducted in the presence of 400 µCi/ml of [alpha -32P]dATP, and DNA reaction products were isolated, treated with Ung/Nfo, and resolved by 0.8% agarose gel electrophoresis as described above. The [32P]DNA was then transferred from the agarose gel to a Gene Screen Plus (PerkinElmer Life Sciences) membrane as described by Koetsier et al. (29). The membrane was dried and used to expose a phosphor screen; 32P-labeled DNA bands were visualized by PhosphorImager using the Scanner Control program (Molecular Dynamics). Form I [32P]DNA (~100 ng) isolated from the BER reactions was treated with excess HinfI restriction endonuclease, and restriction fragments were resolved by 5% nondenaturing polyacrylamide gel electrophoresis (25). After drying the gel, DNA bands were visualized by PhosphorImager, and the relative intensity of various [32P]DNA bands was quantified using the ImageQuant computer program.

Isolation of Repaired Form I DNA-- Form I DNA bands was isolated from agarose gel slices by electroelution using an Elutrap apparatus (Schleicher and Schuell). Electroelution was conducted for 3 h at 150 V in TAE buffer (40 mM Tris acetate, 1 mM EDTA (pH 8.0)), and the recovered form I DNA was concentrated using a Centricon 30 concentrator (Amicon) and buffer-exchanged into distilled water.

Determination of Repair Patch Size-- Standard BER reaction mixtures were prepared as described above except that a 2'-deoxyribonucleoside alpha -thiotriphosphate was used in place of each of the four 2-deoxyribonucleoside triphosphates, and 32P-labeled M13mp2op14 (U·T) form I DNA was used as the BER substrate. The [32P]DNA substrate was constructed as described previously (30, 31) and contained a 32P radiolabel located 13 nucleotides upstream of the target uracil and 7 nucleotides downstream of the SmaI restriction site on the transcribed strand of the lacZalpha gene sequence. Following the BER reactions, DNA products were isolated and resuspended in 20 µl of TE buffer as described above. Samples (4 µl, ~200 ng) were removed for digestion with 10 units of EcoRI for 1 h at 25 °C. The reaction was terminated at 70 °C for 10 min, and samples were incubated in the absence or the presence of various amounts of E. coli exonuclease III (Xth) for 1 h at 37 °C. After incubation, each sample was heated at 70 °C for 10 min, and the [32P]DNA was then cleaved with 10 units of SmaI for 1 h at 25 °C. An equal volume of denaturing formamide dye buffer was added, and [32P]DNA products were resolved by 12% polyacrylamide, 8.3 M urea gel electrophoresis (25). After drying the gel under vacuum, autoradiography was performed, and the amount of 32P radioactivity associated with each band was quantified using a PhosphorImager.

Transfection of E. coli and Determination of Reversion Frequencies-- E. coli NR9162 (mutS) cells were transfected with repaired M13mp2op14 DNA (form I) as described previously (25). Briefly, the transfected cells were diluted into SOB medium, combined with E. coli CSH50 (indicator strain) and top agar containing 0.4 mM isopropyl beta -D-thiogalactopyranoside and 1 mg/ml of 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside, and the mixture was plated on M9 plates. After scoring the phage plaques as either colorless or blue, the reversion frequency was calculated as the ratio of the number of blue plaques to total plaques detected. Secondary screening of each blue plaque was then conducted to purify phage in preparation for nucleotide sequence analysis (25).

Determination of Mutational Spectrum-- An individual blue plaque was picked by touching a P10 pipette tip (Pipetman) to the surface of the plaque so as to extract a miniagarose plug ~0.3 mm long. This material was then injected into an Eppendorf microcentrifuge tube containing 100 µl of PCR mixture (New England Biolabs) comprising 1 unit of Deep Vent DNA polymerase, 1× Thermopol buffer including 2 mM MgCl2, 200 µM dNTPs (Ultrapure; Amersham Pharmacia Biotech), and 100 pmol each of the lacZalpha forward primer (FP-18-mer) and reverse primer (RP-18-mer). PCR was carried out in a Hybaid PCR Express Gradient thermocycler under active temperature control (150 µl of mineral oil in the temperature reference tube) using the following cycling parameters: 94 °C (1 min); 30 cycles of 94 °C (1 min), 45 °C (1 min), and 72 °C (3 min); and 72 °C (10 min). PCR products were purified with a StrataPrep PCR Purification Kit (Stratagene). Approximately 70 µl of 30-70 µg/ml of double-stranded DNA was obtained from a single purified M13 plaque. An aliquot of the purified DNA product (~30 ng) together with 12 pmol of sequencing primer (S-21-mer) complementary to the (+)-strand of M13mp2op14 lacZalpha gene was used for DNA sequence analysis conducted by the Center for Gene Research and Biotechnology (Oregon State University).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Uracil-initiated Base Excision DNA Repair Assay-- An M13mp2 lacZalpha DNA-based reversion assay was utilized to detect uracil-initiated DNA base excision repair and to determine the base substitution error frequency associated with the completed repair reaction (25). Briefly, the arginine codon 14 (CGT) of the lacZalpha gene was replaced with an opal codon (TGA) by site-directed mutagenesis. Heteroduplex DNA (form I) substrate was synthesized containing a U·T base mispair at the first position of the opal codon 14 that served as the uracil target for repair; excision of the uracil residue by uracil-DNA glycosylase initiated the BER pathway (Fig. 1A). Following BER in cell extracts, the M13mp2op14 DNA was recovered and treated in vitro with excess E. coli Ung and Nfo to convert uracil- or AP-site-containing form I DNA to form II molecules. This step effectively removed unreacted and incompletely repaired substrates from the pool of fully repaired form I DNA molecules that were resistant to the Ung/Nfo treatment. The progress of the uracil-initiated BER reaction was monitored by product analysis using agarose gel electrophoresis and ethidium bromide staining (Fig. 1B). As expected, the M13mp2op14 DNA substrate was rapidly converted from form I to form II DNA following uracil excision and incision of the resulting apyrimidinic site. As the DNA repair progressed, form II DNA was reconverted to form I DNA, which was resistant to the Ung/Nfo treatment, in accordance with completed BER.



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Fig. 1.   Scheme for measuring uracil-mediated base excision repair DNA synthesis fidelity. A, M13mp2op14 DNA (form I) containing a site-specific uracil mispaired with thymine located in opal codon 14 at nucleotide position 78 of the lacZalpha gene was constructed as described under "Experimental Procedures." The uracil target for uracil-DNA glycosylase (vertical arrow) and direction of DNA repair synthesis on the (-)-strand (horizontal arrow) resulting from base excision repair are indicated. EcoRI and SmaI endonuclease restriction sites are indicated by vertical lines. B, four standard base excision DNA repair reaction mixtures (100 µl each) were prepared as described under "Experimental Procedures." Reactions were incubated for 0, 15, and 60 min at 30 °C, and the DNA reaction products were recovered and subsequently analyzed by 0.8% agarose gel electrophoresis. In one case, the M13mp2op14 DNA recovered from a 60-min reaction was treated with excess E. coli Ung and Nfo (+) prior to electrophoresis. Form I and II DNA standards (100-12.5 ng) were prepared and resolved on the same agarose gel containing the BER reaction products to quantify the amount (ng) of form I and II DNA (horizontal arrows) produced during BER. C, base substitutions are indicated that lead to reversion of the opal codon 14 and are detected as light and dark blue M13mp2 phage plaques. Only reversion at the opal codon results in a blue plaque phenotype.

The uracil residue in the M13mp2op14 (U·T) DNA substrate was strategically located such that faithful and unfaithful uracil-initiated DNA repair synthesis could be distinguished by the lacZalpha complementation phenotype of the M13mp2 phage genome. If faithful DNA synthesis occurs during BER opposite the template thymine residue at position 78, a dAMP nucleotide will be incorporated into the (-)-strand, and the opal codon will be re-established in both DNA strands. Since the (-)-strand DNA serves as the template for production of single-stranded M13 DNA in E. coli, the resulting phage will be defective in alpha -complementation, and the plaques they produce on the indicator strain will be colorless when grown on medium containing isopropyl beta -D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (X-gal) (Fig. 1C). However, if BER DNA synthesis is unfaithful, dCMP, dGMP, or dTMP may be misincorporated. Each of these base substitutions restores (reverts to wild-type) the alpha -complementation phenotype and produces blue-colored plaques.

Detection of Uracil-initiated BER in E. coli NR8051 (ung+) and NR8052 (ung-) Cell Extracts-- Initial experiments were conducted to detect uracil-initiated BER in E. coli extracts of Ung-proficient (NR8051) and Ung-deficient (NR8052) cells. M13mp2op14 (U·T) DNA (form I) was incubated with each cell-free extract for various times, and then the reaction products were resolved by agarose gel electrophoresis (Fig. 2). In both the Ung-proficient and Ung-deficient cell extracts, a time dependence appearance of repaired form I DNA was observed that indicated completed BER. However, the rate of DNA repair, as determined from the percentage of Ung/Nfo-resistant form I DNA, was much faster in the Ung-proficient strain (Fig. 2A) compared to the Ung-deficient strain (Fig. 2B).



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Fig. 2.   Detection of uracil-mediated BER in E. coli NR8051 (ung+) or NR8052 (ung-) cell extracts. Standard BER reaction mixtures (100 µl) containing 1 µg of M13mp2op14 (U·T) DNA or 0.1 mg of E. coli NR8051 (A) or NR8052 (B) cell extracts were incubated for 0, 10, 20, 30, 40, and 60 min at 30 °C (lanes 1-6, respectively). Each reaction was then terminated by the addition of 25 µl of 0.1 M EDTA, and the samples were heated at 70 °C for 3 min. The M13mp2op14 DNA was recovered following the BER reactions, subjected to Ung/Nfo treatment, and analyzed by 0.8% agarose gel electrophoresis as described under "Experimental Procedures." As a control, M13mp2op14 (U·T) DNA (1 µg) was mock-reacted and then treated Ung and Nfo (lane C). Untreated M13mp2op14 (U·T) DNA (100 ng) and a sample containing 1 µg of a 1-kilobase pair DNA ladder (Life Technologies, Inc.) were employed as reference standards (lanes S and M, respectively). The arrows indicate the location of form I and II DNA bands detected by ethidium bromide staining.

Impact of Ugi on Uracil-initiated BER-- To further examine the observation that uracil-initiated BER occurred in Ung-deficient cell extracts, we utilized the Ugi protein to inactivate E. coli uracil-DNA glycosylase. BER reactions were performed in extracts of E. coli NR8051 and NR8052 cells in the absence or presence of excess Ugi. A time course of repair was conducted, and the amount of repaired DNA (form I) detected relative to form II DNA was ascertained (Fig. 3). The results showed quantitatively that during the first 20 min of the reaction, the initial rate of repair was ~5.5-fold slower in E. coli NR8052 (ung-1) compared with the NR8051 cell extract. As expected, the addition of Ugi did not appear to affect the rate or extent of repair in extracts of NR8052 (ung-1). However, uracil-initiated BER was substantially diminished in extracts of the Ung-proficient strain NR8051 when supplemented with Ugi. The level of repaired DNA, detected after 60 min, in the Ugi-supplemented Ung-proficient cell extract was comparable with that observed in both the Ung-deficient and Ugi-supplemented Ung-deficient cell extracts.



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Fig. 3.   Analysis of E. coli uracil-mediated BER in the presence and absence of Ugi. Standard BER reaction mixtures (100 µl) containing 1 µg of M13mp2op14 (U·T) DNA and 0.1 mg of E. coli NR8051 or NR8052 cell extracts were prepared as described in Fig. 2, except that Ugi (1000 units) was added in some cases. Reactions were incubated at 30 °C for the times indicated and then processed as described under "Experimental Procedures." The M13mp2op14 substrate DNA was isolated, treated with E. coli Ung and Nfo, and analyzed by 0.8% agarose gel electrophoresis. DNA bands detected by ethidium bromide staining were quantitatively measured using a gel documentation system, and the percentage of form I DNA in each sample was determined. The amount of repaired DNA detected was plotted as percentage of form I DNA for the following reactions: E. coli NR8051 minus Ugi (), NR8051 plus Ugi (open circle ), NR8052 minus Ugi (black-square), and NR8052 plus Ugi (). Mean values and S.D. of three experiments are represented.

Evidence for Uracil-mediated BER DNA Synthesis-- To determine whether uracil-initiated BER DNA synthesis was involved in the production of Ung/Nfo-resistant form I DNA, standard BER reactions were conducted in the presence of [alpha -32P]dATP. After agarose gel electrophoresis of the BER reaction products, examination of the phosphor images showed the incorporation of [32P]dAMP into the Ung/Nfo-resistant form I DNA recovered from reactions containing cell extracts of E. coli NR8051 (ung+) (Fig. 4A) as well as NR8052 (ung-) (Fig. 4B). Notably, the intensity of form I [32P]DNA generated in the Ung-proficient cell extract was considerably greater than that produced in the Ung-deficient extract, although the amount of 32P radioactivity incorporated into the total DNA (form I plus form II) in each reaction was similar. Quantification of the 32P radioactivity associated with the form I DNA band revealed that the amount of DNA synthesis generated in Ung-deficient cell extracts was ~10-fold less than that produced in Ung-proficient extracts after 60 min (Fig. 4C). When taken together with the findings in Fig. 3, the results suggest that the reduced level of DNA synthesis was the likely result of an overall reduction in the level of BER.



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Fig. 4.   Incorporation of [alpha -32P]dAMP into M13mp2op14 DNA during uracil-initiated BER. Two sets of standard BER reaction mixtures (100 µl) containing 1 µg of M13mp2op14 (U·T) DNA, 40 µCi of [32P]dATP, and 0.1 mg of E. coli NR8051 (A) or NR8052 (B) cell extracts were incubated for 0, 5, 10, 30, and 60 min at 30 °C (lanes 1-5, respectively). The reactions were terminated; M13mp2op14 [32P]DNA was isolated, treated with E. coli Ung and Nfo, and analyzed by 0.8% agarose gel electrophoresis; and the [32P]DNA fragments were blotted from the agarose gel to a Gene Screen Plus membrane as described under "Experimental Procedures." The arrows indicate the location of form I and II DNA bands visualized by PhosphorImager (Molecular Dynamics). C, the relative amount of [32P]dAMP incorporated into repaired form I DNA was determined for reactions containing NR8051 (black-square) and NR8052 () cell extracts and plotted after subtracting background values. The results shown represent the mean and S.D. of three experiments.

Specificity of Uracil-mediated DNA Repair Synthesis in E. coli Ung-proficient Cell Extracts-- To determine the distribution of [32P]dAMP incorporation associated with the repaired DNA molecules, [32P]DNA isolated from the BER reactions described in Fig. 4 was subjected to HinfI restriction endonuclease digestion and resolved by nondenaturing polyacrylamide gel electrophoresis (Fig. 5). While there are 26 HinfI recognition sites in the M13mp2op14 DNA sequence, HinfI digestion produces only 16 DNA fragments in excess of 200 bp. Among these, the 529-bp fragment containing the uracil site is bordered by 262- and 253-bp fragments positioned 5' and 3', respectively (Fig. 5A). Following electrophoresis, inspection of the phosphor image showed that [32P]dAMP incorporation occurred preferentially in the 529-bp fragment in a time-dependent manner (Fig. 5, B and C). The minor amount of incorporation detected on the neighboring fragments (262 and 253 bp) was assumed to correspond to nonspecific background, since a similar level of incorporation was observed associated with a 486-bp fragment located on the opposite side of the M13mp2op14 DNA molecule (Fig. 5C).



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Fig. 5.   Specificity of BER DNA synthesis in E. coli NR8051 cell extracts. A, HinfI restriction map of M13mp2op14 DNA indicating restriction sites (hash marks) and the location of the 253-, 529-, 261-, and 486-bp DNA fragments. The uracil (U) residue targeted for base excision repair is located at position 78 in the (-)-strand of the lacZalpha gene. B, after conducting standard BER reactions containing E. coli NR8051 cell extracts, DNA was isolated at various times, as described under "Experimental Procedures." DNA samples obtained from reactions conducted for 0, 5, 10, 30, and 60 min (lanes 1-5, respectively) were subjected to digestion with 10 units of HinfI for 1 h at 37 °C and resolved by 5% nondenaturing polyacrylamide gel electrophoresis. The location of the DNA fragment (U-529) that contained the site-specific uracil is indicated by an arrow, as are the locations of three other fragments. C, [32P]dAMP incorporation into the DNA fragments of 253 (striped bar), 529 (black bar), 261 (white bar), and 486 bp (stippled bar) was determined using a PhosphorImager and ImageQuant software (Molecular Dynamics). The relative intensity of each DNA fragment was measured at the time point indicated after subtracting background values. Error bars represent the S.D. of three experiments.

Uracil-dependent BER DNA Synthesis-- To determine whether the preferential incorporation of [32P]dAMP into the 529-bp fragment observed in Fig. 5 was the result of BER DNA repair synthesis instigated by uracil excision, standard BER reactions were conducted as before, but the M13mp2op14 DNA substrate contained either a U·T or A·T base pair at the first position of the lacZalpha opal codon 14. After incubation for 1 h, the reaction products were recovered, and HinfI [32P]DNA fragments were resolved by polyacrylamide gel electrophoresis (Fig. 6). Inspection of the phosphor image showed that preferential incorporation of [32P]dAMP into the 529-bp fragment of the (U·T) DNA occurred in both the E. coli NR8051 and NR8052 cell extracts relative to [32P]dAMP incorporation into the same 529-bp fragment of the (A·T) DNA. The results indicated that a uracil residue located in the target DNA fragment stimulated DNA synthesis by 17.2- and 5.8-fold above background for the reactions containing E. coli NR8051 and NR8052 cell extracts, respectively.



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Fig. 6.   Uracil-dependent BER DNA synthesis in E. coli NR8051 and NR8052 cell extracts. Standard BER reaction mixtures (100 µl) containing 1 µg of M13mp2op14 (A·T) DNA (lanes 1 and 3) or (U·T) DNA (lanes 2 and 4) were incubated with 0.1 mg of E. coli NR8051 (lanes 1 and 2) or NR8052 (lanes 3 and 4) cell extract protein in the presence of 40 µCi of [32P]dATP. After incubation for 1 h at 30 °C, the DNA reaction products were isolated and subjected to HinfI restriction endonuclease digestion, and the [32P]DNA fragments were then analyzed as described in Fig. 5. The location of the 253-, U-529-, 261-, and 486-bp HinfI DNA fragments is indicated by arrows.

Uracil-initiated BER in Cell Extracts of E. coli Defective in ung and dug-- To further assess the role of the dug gene product in uracil-initiated BER, repair reactions were conducted using the three E. coli isogenic strains BH156 (ung- dug+), BH157 (ung+ dug-), and BH158 (ung- dug-). Examination of the uracil-initiated BER reaction time course by agarose gel electrophoresis revealed a time-dependent accumulation of Ung/Nfo-resistant form I DNA in both the E. coli BH156 and BH157 cell extracts (Fig. 7). However, the rate of repair appeared to be 5-fold greater in the reactions containing E. coli BH157 cell extract. In contrast, Ung/Nfo-resistant form I DNA was not observed in BER reactions with BH158 cell extracts (Fig. 7, lanes 13-18). This experiment indicated that uracil-mediated BER occurred in the absence of either Ung or Dug but at different rates.



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Fig. 7.   Analysis of uracil-mediated BER in E. coli BH156 (ung), BH157 (dug), and BH158 (ung, dug) cell extracts. Three sets of standard BER reaction mixtures (100 µl) containing 1 µg of M13mp2op14 (U·T) DNA were incubated for 0, 5, 10, 20, 30, and 60 min at 30 °C with 0.1 mg of cell extract protein from E. coli BH156 (lanes 1-6, respectively), BH157 (lanes 7-12, respectively), or BH158 (lanes 13-18, respectively). Form I DNA reaction products were isolated, treated with E. coli Ung and Nfo, and resolved by 0.8% agarose gel electrophoresis (inset) as described under "Experimental Procedures." Untreated M13mp2op14 (U·T) DNA (100 ng) was used as a reference standard (lanes S). As a control, M13mp2op14 (U·T) DNA (1 µg) was mock-reacted, isolated, and then subjected to Ung/Nfo treatment (lane C). The location of form I and II DNA bands is indicated by arrows. The amount of form I and II DNA detected by ethidium bromide staining was quantitatively measured with a gel documentation system, and the percentage of form I DNA was determined. The results of two independent experiments are plotted for E. coli BH156 (), BH157 (black-square), and BH158 (black-triangle).

Uracil-initiated BER Patch Size-- We utilized the approach developed by Huang et al. (32) and Gish and Eckstein (33) and modified as described previously (30, 31) to determine the patch size of DNA repair synthesis associated with uracil-mediated BER using the M13mp2op14 DNA substrate (Fig. 8). This approach relies on the incorporation of 2'-deoxyribonucleoside alpha -thiomonophosphates during the BER reaction to render the repaired DNA strand resistant to in vitro digestion with E. coli exonuclease III. As illustrated in Fig. 1A, the uracil target site in M13mp2op14 DNA was located on the (-)-strand, 20 nucleotides upstream (5'-side) of a unique EcoRI site and 19 nucleotides downstream (3'-side) of a unique SmaI site. For the purpose of measuring the repair patch size, a site-specific [32P]dCMP residue was introduced between the uracil target and the SmaI site, at nucleotide position 90 in the (-)-strand DNA. Two reference standards were created by treating the M13mp2op14 [32P]DNA substrate with EcoRI and SmaI alone (Fig. 8A, lanes 1 and 13) or in conjunction with Ung and Nfo (Fig. 8A, lanes 2 and 12), which produced the expected 32P-labeled fragments of 40 and 19 nucleotides, respectively. The 19-mer corresponded to the BER intermediate formed immediately prior to DNA synthesis and defined the 5'-boundary of the repair patch (30, 31). To locate the 3'-boundary of DNA synthesis produced during ung-proficient and ung-deficient BER, recovered M13mp2op14 [32P]DNA was examined from BER reactions conducted with E. coli NR8051, NR8052, and mock treatments. In each case, the [32P]DNA was linearized with EcoRI and then digested in the 3'-5' direction with excess exonuclease III. Exonuclease III digestion was expected to terminate upon encountering the first phosphorothiol linkage (34); accordingly, this ultimate dNMP[alpha S] residue defined the 3'-boundary of the repair patch. Subsequent cleavage with SmaI produced a [32P]DNA fragment, the length of which indicated the BER patch size (i.e. 20-, 21-, and 22-mers corresponded to a repair tract of 1, 2, and 3 nucleotides, respectively). As anticipated, exonuclease III digestion of the mock-treated [32P]DNA (Fig. 8A, lanes 4 and 5) did not produce detectable fragments smaller than the 40-mer control (Fig. 8A, lane 3), since BER had not occurred and dNMP[alpha S]s were not incorporated into the [32P]DNA substrate. In contrast, discrete fragments were generated following exonuclease III digestion of repaired M13mp2op14 [32P]DNA produced in BER reactions containing E. coli NR8051 cell extracts (Fig. 8A, lanes 6-8) and NR8052 (Fig. 8A, lanes 9-11). The amount of each 32P-labeled DNA fragment was quantitatively determined, and the distribution of the repair patch size was plotted in Fig. 8B. In both cell extracts, the vast majority of BER occurred via a long patch mechanism, whereas short patch (1-nucleotide) repair accounted for ~7% of the BER events. While the repair patch size distribution was similar in each reaction, the ung-proficient BER reaction was biased toward longer repair patches.



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Fig. 8.   Analysis of DNA repair patch size associated with uracil-mediated BER reactions. A, standard BER reaction mixtures (100 µl) containing 1 µg of M13mp2op14 (U·T) [32P]DNA; a 20 µM concentration each of dATP[alpha S], dTTP[alpha S], dGTP[alpha S], and dCTP[alpha S]; and 0.1 mg of cell extract protein of E. coli NR8051 (lanes 6-8) and NR8052 (lanes 9-11) were incubated for 60 min at 30 °C. As a control, M13mp2op14 (U·T) [32P]DNA (1 µg) was mock-reacted in the absence of cell extract protein (lanes 3-5). DNA products were isolated, samples (~200 ng) were digested with EcoRI and then incubated with 0 (lanes 3, 6, and 9), 2 (lanes 4, 7, and 10), and 20 (lanes 5, 8, and 11) units of E. coli exonuclease III. Following exonuclease III digestion, the DNA was cleaved with SmaI and then resolved by 12% polyacrylamide, 8.3 M urea gel electrophoresis as described under "Experimental Procedures." The DNA size markers, 40-mer (lanes 1 and 13) generated by digesting 200 ng of M13mp2op14 (U·T) [32P]DNA with EcoRI and SmaI, and the 19-mer (lanes 2 and 12) produced by additional treatment with Ung and Nfo, are indicated by arrows. B, the amount of 32P radioactivity detected in each band in A was quantitatively measured using a PhosphorImager, and the results for the E. coli NR8051 (white bars) and NR8052 (black bars) reactions digested with 20 units of E. coli exonuclease III are plotted. The [32P]DNA bands of 20-40 nucleotides in length corresponded to BER repair patches of 1-21 nucleotides in length, respectively. The relative amount of 32P label in each band (% Distribution) was determined by dividing the amount of 32P radioactivity detected per band by the total 32P signal detected for all bands and multiplying by 100. Mean values and S.D. for the distribution of four experiments are indicated.

Error Frequency Associated with Uracil-initiated BER-- The M13mp2op14 lacZalpha DNA-based reversion assay was utilized to ascertain the frequency of mutations produced during uracil-initiated BER (Table I). Initially, the background reversion frequency of the M13mp2op14 (A·T) DNA was determined in extracts of E. coli NR8051 and NR8052 cells. In each case, a similar reversion frequency, 0.14 × 10-4 (NR8051) and 0.21 × 10-4 (NR8052), was observed. Next, the reversion frequency associated with uracil-mediated BER of M13mp2op14 (U·T) DNA was determined to be 5.5 × 10-4 and 19.7 × 10-4 for reactions conducted with E. coli NR8051 and NR8052 cell extracts, respectively (Table I). Thus, the absence of ung promoted an increase (~3.6-fold) in the reversion frequency. Similar results were obtained when BER of M13mp2op14 (U·T) DNA was performed using the analogous cell extracts (NR80511 and NR80521) that contained a mutS mutation. Therefore, methyl-directed mismatch repair did not seem to influence the fidelity of the BER reaction.


                              
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Table I
Frequency of mutations produced by uracil-initiated BER in E. coli NR8051 and NR8052 cell-free extracts
Standard BER reaction mixtures (500 µl) were prepared that contained 0.5 mg of E. coli NR8051, NR80511 mutS, NR8052, or NR80521 mutS cell extract and 5 µg of M13mp2op14 (U · T) or (A · T) DNA. After incubation at 30 °C for 60 min, the reactions were terminated, DNA products were recovered, and form I DNA resistant to Ung/Nfo treatment was isolated by 0.8% agarose gel electrophoresis as described under "Experimental Procedures." E. coli NR9162 cells were then transfected with the form I DNA, and the M13mp2 lacZalpha DNA-based reversion assay was performed as described under "Experimental Procedures."

In an effort to clarify the role of Dug as a potential mediator of the elevated error frequency-associated Ung-deficient uracil-initiated BER, repair reactions containing cell extracts of E. coli NR8051 were supplemented with Ugi or Dug protein, and reactions containing NR8052 cell extracts were supplemented with Ugi or Ung protein. Following the recovery of repaired DNA, the reversion frequency of the M13mp2op14 (U·T) DNA substrate was determined (Table II). Supplementation of NR8051 extracts with Ugi gave rise to a reversion frequency (40.3 × 10-4) elevated ~7-fold relative to that measured for NR8051 extracts alone (5.5 × 10-4). Conversely, when extracts of NR8052 were supplemented with Ung, the reversion frequency was reduced ~8-fold relative to that obtained for NR8052 extracts alone. Taken together, these results suggest that uracil-mediated BER conducted in the absence of Ung was more mutagenic than Ung-initiated BER. Consistent with this interpretation, the addition of Dug to NR8051 extracts resulted in an increase (~2-fold) in the frequency of opal codon 14 reversion, whereas the addition of Ugi to NR8052 extracts did not appreciably augment the elevated mutation frequency.


                              
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Table II
Frequency of mutations produced by uracil-initiated BER in E. coli NR8051 and E. coli NR8052 cell-free extracts supplemented with purified Ung, Dug, or Ugi protein
Standard BER reaction mixtures (500 µl) were prepared that contained 5 µg of M13mp2op14 (U · T) DNA and 0.5 mg of E. coli cell extract, as indicated. After incubation at 30 °C for 60 min, the reactions were terminated, DNA products were recovered, and Ung/Nfo-resistant form I DNA was isolated by 0.8% agarose gel electrophoresis as described under "Experimental Procedures." The form I DNA was then used to transfect E. coli NR9162 cells, and the M13mp2 lacZalpha DNA-based reversion assay was performed as described under "Experimental Procedures."

The reversion frequency of uracil-initiated BER at opal codon 14 was also determined for E. coli of a different genetic background (Table II). Experiments conducted with E. coli BH156 (ung- dug+) exhibited a reversion frequency of 25.9 × 10-4. This value was similar to those obtained for E. coli NR8052 (19.7 × 10-4), NR8052 supplemented with Ugi (39.4 × 10-4), and NR8051 supplemented with Ugi (40.3 × 10-4). In the complementary experiment, uracil-initiated BER in extracts of E. coli BH157 (ung+ dug-) resulted in a relatively low reversion frequency of 5.7 × 10-4, which compared favorably with that obtained for extracts of NR8051 (5.5 × 10-4). Determination of the reversion frequency associated with the E. coli BH158 (ung- dug-) was not possible, since the production of Ung/Nfo-resistant form I DNA was not detected (Fig. 7, lanes 13-18).

Mutational Spectra-- DNA from individual revertant M13mp2op14 phage plaques was amplified, and the nucleotide sequence of the lacZalpha gene encompassing the opal codon 14 reversion target was determined to define the nature and the distribution of base substitutions introduced during the process of BER. First, the spontaneous mutational spectrum was determined for M13mp2op14 (A·T) DNA obtained from the BER reactions containing E. coli NR8051 cell extracts. Inspection of the mutational distribution showed that of the 30 mutants sequenced, 20 (67%) reverted by base substitutions in the third nucleotide position of the opal codon (Fig. 9A). Of these 20, 12 were A to C transversions, and eight were A to G transition mutations. A relatively minor class of mutations occurred in the first position. Eight mutations were detected; seven of these were T to A transversions. A similar bias for mutations at the third position was also observed for M13mp2op14 (A·T) DNA incubated in a BER reaction containing extracts of NR8052 (data not shown). Second, the mutational spectrum of uracil-initiated BER, obtained using the M13mp2op14 (U·T) DNA substrate and E. coli NR8051 cell extract, revealed that 80 of the 82 mutants sequenced reverted at the first nucleotide position of the opal codon (Fig. 9B); thus, these mutations occurred almost exclusively at the uracil target site. The large majority of these uracil-initiated BER mutations were T to G transversions (70%) and T to A transversions (29%). Third, the mutational spectrum of uracil-initiated BER in E. coli NR8051 and NR8052 cell extracts supplemented with Ugi was compared using the M13mp2op14 (U·T) DNA substrate. From the BER reaction containing NR8051 cell extracts, 90 revertants were analyzed, and 98% (88 of 90) of the base substitutions occurred in the first nucleotide position (Fig. 9C). The majority of these mutations (73%) were determined to be T to G transversions (64 of 88); T to A transversions accounted for the remainder. The spectra of mutations obtained from the BER reaction containing NR8052 extracts also consisted almost exclusively of first nucleotide base substitution mutations (94 out of 95) (Fig. 9D). Of these, 60% (56 of 94) were T to G transversions, while 40% were T to A transversions.



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Fig. 9.   E. coli mutational spectrum of uracil-initiated base excision repair. Four standard BER reactions were performed using either E. coli NR8051 (A, B, and C) or NR8052 (D) cell extracts and M13mp2op14 DNA as described in Table I. One reaction contained M13mp2op14 (A·T) DNA (A), and the other three reactions were prepared with the (U·T) DNA substrate (B-D), while 1000 units of Ugi were added to reactions in C and D. Following transfection of NR9162 cells with Ung/Nfo-resistant form I DNA recovered from BER reactions, blue plaques were isolated, phage were subjected to PCR-mediated lacZalpha DNA amplification, and DNA sequence analysis was conducted as described under "Experimental Procedures." The nucleotide sequence (TGA) of the opal codon 14 in the template DNA strand used for uracil-initiated BER DNA synthesis is indicated. The four possible deoxyribonucleoside triphosphates used for incorporation into the primer strand are indicated with the corresponding coded amino acid (parenthesis). For each BER reaction, the number of base substitution mutations detected by DNA sequence analysis is plotted.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have examined the ability of various E. coli cell extracts to carry out uracil-initiated BER of a covalently closed circular M13mp2 lacZalpha DNA substrate for the purpose of determining the fidelity associated with complete BER reactions. To our knowledge, this report is the first to present fidelity measurements of BER associated with Ung-proficient or Ung-deficient E. coli cells. Several previous studies have implicated Dug as participating in uracil-DNA repair in Ung-deficient cells (14, 16, 22); however, a recent report concluded that Dug plays no role in uracil-mediated BER (35). The results presented here support the proposition that Dug can participate in uracil-DNA repair and provide additional evidence that Dug is primarily responsible for uracil-mediated BER in the absence of Ung. These findings reinforce the observations of Gallinari and Jiricny (22), who originally identified the double-stranded uracil-DNA glycosylase activity in cell-free extracts of E. coli NR8052 that carried the ung-1 mutation; however, they do not exclude the possibility that Dug may also play a role in the repair of 3,N4-ethenocytosine residues, as has been suggested by other investigators (16, 35).

Several lines of evidence support the interpretation that the uracil-mediated repair observed in this study occurred via a BER pathway. First, we observed that upon conclusion of the BER reaction, form I DNA was generated that was resistant to cleavage by the combined treatment of Ung and Nfo. This finding indicated that all steps of uracil-DNA repair had been completed. Second, we demonstrated that DNA synthesis occurred preferentially in the HinfI DNA fragment (529 bp) encompassing the uracil target. Furthermore, DNA synthesis within the 529-bp fragment was almost exclusively dependent on the presence of a uracil residue. Third, the addition of Ugi to cell extracts of E. coli NR8051 substantially inhibited the formation of Ung/Nfo-resistant form I DNA. However, complete inhibition was not observed, since E. coli NR8051 is proficient for Dug activity, which is insensitive to inhibition by Ugi (14). Fourth, DNA synthesis was associated with a repair patch involving <= 20 nucleotides and was oriented 3' to the uracil target. These results were not characteristic of the E. coli methyl-directed DNA mismatch repair pathway, where DNA repair synthesis tracts of 1 kilobase pair or more can occur (36). Last, formation of Ung/Nfo-resistant form I DNA was not observed in BER reactions containing extracts of E. coli BH158, in which both ung and dug are inactivated. This observation strongly suggests that Ung and Dug are the predominant, if not exclusive, uracil excision activities in wild type E. coli, and that BER was initiated by one or the other uracil-DNA glycosylase.

Examination of the kinetics of BER in extracts of NR8051 (ung+) cells showed that 60% of the M13mp2op14 (U·T) DNA substrate was repaired after a 20-min reaction. In contrast, the rate of BER in extracts of NR8052 (ung-) cells was ~5.5-fold lower. One interpretation of these data is that Ung rapidly turns over during BER, whereas Dug has a low rate of turnover. Consistent with this interpretation is the observation of Sung and Mosbaugh (14, 17) that the addition of purified Dug to the Ung-deficient reaction led to an increase in the rate of repair early in the reaction time course. Since strong binding by Dug to its reaction product AP site·G DNA has been demonstrated (14), it is tempting to speculate that Dug binding hinders efficient processing of the abasic site, impeding completion of the BER pathway. While E. coli endonuclease IV was shown to stimulate the catalytic turnover of Dug, Dug-mediated BER remained significantly less than Ung-initiated BER, even under the stimulated condition (14, 17). Thus, the role of Dug in uracil-DNA repair conducted via the BER pathway may be to provide an auxiliary repair system that serves as a secondary line of defense against uracil-provoked mutagenesis.

What influence does the fidelity of BER have on uracil-initiated mutagenesis in E. coli? Based on the results reported in Table I, the error frequency associated with Ung-mediated BER in cell-free extracts was determined to be 5.5 × 10-4 per repaired uracil residue. Under normal conditions, the vast majority of uracil in E. coli DNA results from dUMP incorporation during replication (1, 3). Tye et al. (3) reported that 1 uracil residue was introduced per 1200 nucleotides polymerized. Accordingly, one would anticipate that ~4000 uracil residues are incorporated per round of chromosomal DNA replication (1). Based on these reports, we extrapolate that E. coli Ung-proficient BER could generate approximately two mutations per cycle of semiconservative DNA replication, providing that error correction did not occur prior to mutation fixation. The addition of Ugi to the Ung-proficient BER reactions resulted in a ~7-fold increase in reversion frequency without an accompanying change in the mutational specificity. Thus, Ugi produced an ung phenotype that reflected the elevated reversion frequency and mutational specificity associated with Dug-mediated BER. The ung mutator phenotype was reproduced in strains sharing the E. coli GM31 genetic background, namely BH156 and BH157. Taken together, these results indicate that Ung-mediated BER occurs with higher fidelity than that initiated by Dug.

The mutational specificity of E. coli uracil-initiated BER repair observed in the opal codon 14 TGA reversion assay appears distinct from that observed in other fidelity assays conducted with purified E. coli DNA polymerase I (large fragment). In extracts of Ung-proficient E. coli cells (NR8051), our mutational analysis revealed that T to G transversions, resulting presumably from T·C mispairs, were dominant (56 of 79), while T to A transversions, the likely result of T·T mispairs, comprised the remainder (23 of 79). In contrast, Minnick et al. (37), using a 361-base gap-filling TGA reversion assay and 3' to 5' exonuclease-deficient DNA polymerase I (large fragment), observed that the error rate of dGTP incorporation opposite T was ~49-fold greater than that of dCTP incorporation opposite T and ~10-fold greater than dTTP incorporation opposite T. Perhaps it is not surprising that our results differ from those reported by Minnick et al. (37), since we have utilized a system in which all steps of the BER pathway are represented. The mutational specificity of uracil-mediated BER is the end result of DNA repair synthesis, which includes misincorporation, proofreading, and/or misextension, and must be followed by ligation. On the other hand, the 361-base gap-filling assay is restricted to measurement of the accuracy of the polymerization step in the absence of competing reactions and does not require ligation.

We examined the patch size of BER DNA synthesis associated with Ung- versus Dug-mediated repair. In both cases, the patch size was heterogeneous, ranging from 1 to ~20 nucleotides in length, although the size of the repair patch produced in Dug-mediated BER reactions was consistently somewhat shorter. Quantification of the distribution of the repair patches showed that the mean patch size was 11 nucleotides in Ung-mediated BER compared with 7 nucleotides in Dug-mediated BER. A small amount (~7%) of 1-nucleotide replacement synthesis was observed in both systems; however, the predominant type of DNA repair synthesis was long patch. The latter observation is consistent with the results of Sandigursky et al. (23), who found that repair of a U·G base pair in a closed circular plasmid involved replacement of ~15 nucleotides downstream of the uracil target. The first experiments conducted to elucidate the repair patch size associated with BER in E. coli cell extracts utilized a duplex oligodeoxynucleotide 30-mer DNA with a single U·G base pair located approximately in the middle of the substrate (13). Interestingly, under these conditions, more than 70% of DNA repair synthesis involved incorporation of a single nucleotide (13). As previously pointed out, the size of the repair patch may be influenced by the nature of the DNA repair substrate (23, 38). Thus, short oligodeoxynucleotide substrates may not provide a platform sufficient for interaction with DNA polymerase and accessory repair proteins.

It is generally accepted that DNA polymerase I occupies the primary role in uracil-mediated DNA repair synthesis in E. coli (39, 40). Our analysis of the mutational spectra derived from Ung-proficient and Ung-deficient extracts suggests that the specificity of misinsertion remains essentially the same regardless of the uracil-DNA glycosylase involved; accordingly, one might infer that the same DNA polymerase is involved in Ugi-resistant as well as in Ugi-sensitive uracil-DNA repair. Given the relatively high mutation frequencies we observed in this study, a role for the newly discovered polymerase IV and/or polymerase V DNA polymerases in BER cannot be formally excluded. These DNA polymerases have been described as low fidelity enzymes that exhibit error rates of ~10-3 to 5 × 10-4 when copying undamaged DNA in vitro (41); however, experiments to assess the contribution of these enzymes to BER have not yet been carried out. The molecular mechanisms underlying the mutational specificity of uracil-initiated BER in E. coli and the increased reversion frequency associated with the Dug-mediated BER pathway await further elucidation.


    ACKNOWLEDGEMENT

We thank Nanci Adair for conducting the DNA sequence analysis at the Center for Gene Research and Biotechnology.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM32823 and ES00210. This is Technical Report 11708 from the Oregon Agricultural Experiment Station.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. Tel.: 541-737-1797; Fax: 541-737-0497; E-mail: mosbaugd@ucs.orst.edu.

Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005894200

2 Base pairs and mispairs are described by listing the base in the repaired strand first and then the base in the template strand.

3 No conversion of form I M13mp2op14 (A·T) DNA to form II following Ung/Nfo treatment was detected, indicating that this DNA preparation did not support uracil-initiated BER.


    ABBREVIATIONS

The abbreviations used are: BER, base excision repair; AP, apurinic/apyrimidinic; dRP, deoxyribose 5'-phosphate; PCR, polymerase chain reaction; bp, base pair(s); dNMP[alpha S], 2'-deoxyribonucleoside alpha - thiotriphosphate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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