From the Departments of 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
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ABSTRACT |
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The error frequency and mutational specificity
associated with Escherichia coli uracil-initiated base
excision repair were measured using an M13mp2 lacZ 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 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.
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 lacZ 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 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
[ 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 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
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
lacZ Uracil-initiated Base Excision DNA Repair Assay--
An M13mp2
lacZ
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
lacZ Detection of Uracil-initiated BER in E. coli NR8051
(ung+) and NR8052 (ung 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.
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 [ 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).
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
lacZ 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 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 Error Frequency Associated with Uracil-initiated BER--
The
M13mp2op14 lacZ
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
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 Mutational Spectra--
DNA from individual revertant M13mp2op14
phage plaques was amplified, and the nucleotide sequence of the
lacZ We have examined the ability of various E. coli cell
extracts to carry out uracil-initiated BER of a covalently closed
circular M13mp2 lacZ 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 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 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 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
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
-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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
-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 [
-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).
-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.
-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 lacZ
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.
-D-thiogalactopyranoside and 1 mg/ml of
5-bromo-4-chloro-3-indolyl
-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).
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
lacZ
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
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
lacZ
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
lacZ 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.
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
-complementation, and the plaques they produce on the indicator
strain will be colorless when grown on medium containing isopropyl
-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl
-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
-complementation phenotype and produces blue-colored plaques.
) 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.
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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 (
), NR8052 minus Ugi (
), and NR8052 plus
Ugi (
). Mean values and S.D. of three experiments are
represented.
-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
[ -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 (
) and NR8052 (
) cell
extracts and plotted after subtracting background values. The results
shown represent the mean and S.D. of three experiments.
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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 lacZ
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.
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.
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
(
), and BH158 (
).
-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[
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[
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[ S], dTTP[
S], dGTP[
S], and dCTP[
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.
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.
Frequency of mutations produced by uracil-initiated BER in E. coli
NR8051 and NR8052 cell-free extracts
DNA-based reversion assay was
performed as described under "Experimental Procedures."
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.
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
DNA-based reversion assay was
performed as described under "Experimental Procedures."
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).
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 lacZ
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
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).
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.
)
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.
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.
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.
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ACKNOWLEDGEMENT |
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We thank Nanci Adair for conducting the DNA sequence analysis at the Center for Gene Research and Biotechnology.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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The abbreviations used are:
BER, base excision
repair;
AP, apurinic/apyrimidinic;
dRP, deoxyribose 5'-phosphate;
PCR, polymerase chain reaction;
bp, base pair(s);
dNMP[S], 2'-deoxyribonucleoside
- thiotriphosphate.
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