THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 274, No. 43, Issue of October 22, pp. 31034 –31038, 1999
Printed in U.S.A.
The Role of the Escherichia coli Mug Protein in the Removal of
Uracil and 3,N4-Ethenocytosine from DNA*
(Received for publication, April 27, 1999, and in revised form, July 28, 1999)
Eugene Lutsenko and Ashok S. Bhagwat‡
From the Department of Chemistry, Wayne State University, Detroit, Michigan 48202
Cytosine is the most unstable of the four bases in DNA and
deaminates hydrolytically to create UzG mismatches. If unrepaired, uracil can pair with an adenine during replication causing a C to T mutation. For this reason, cells contain uracil-DNA
glycosylase (Ung), an enzyme that removes the uracil and
initiates its replacement with cytosine. The importance of Ung
in mutation avoidance is evidenced by the observation that ung
strains of Escherichia coli (1) and yeast (2) accumulate C to T
mutations.
Cytosines methylated at position 5 similarly deaminate to
create TzG mismatches, which are not subject to repair by Ung.
In E. coli, a specialized mismatch correction process called very
short patch repair corrects these mispairs to CzG (3). The key
enzyme in this repair pathway is a sequence-specific, mis-
* The work presented here was supported by National Institutes of
Health Grant GM53273. The costs of publication of this article were
defrayed in part by the payment of page charges. This 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: 463 Chemistry,
Dept. of Chemistry, Wayne State University, Detroit, MI 48202. Tel.:
313-577-2547; Fax: 313-577-8822; E-mail: [email protected].
match-specific endonuclease, Vsr, which hydrolyzes the phosphodiester linkage preceding the mismatched T (4). No eukaryotic sequence homologs of this enzyme have been reported;
instead a DNA glycosylase is thought to serve the same function (5). This enzyme excises thymines from TzG mismatches
(6) and prefers mismatches that are followed by a GzC pairs (7,
8). This enzyme, thymine-DNA glycosylase (TDG),1 could prevent mutations when 5-methylcytosines within CG dinucleotides deaminate to thymine.
The cDNA for TDG was cloned and its sequence was determined (9). Remarkably, a sequence homolog of this protein was
found in E. coli and Serratia marcescence (10). The investigators who made this observation suggested that the bacterial
homolog was a uracil-DNA glycosylase specific for UzG mismatches and named it mismatch-specific uracil-DNA glycosylase (Mug). Their conclusions were based on properties of truncated forms of TDG, and biochemical assays done using E. coli
cell-free extracts. They further suggested that it may act as a
backup enzyme for Ung and may be important in avoiding
mutations during stationary phase of cell growth (10).
Because there was no evidence in previous work by any
research group that such a backup enzyme existed, we tested
this possibility. For this purpose, we studied the repair of UzG
and TzG mismatches in mug1 and mug strains. Our results
clearly indicate that Mug plays no role in the repair of UzG or
TzG mismatches and may repair 3,N4-ethenocytosinezG mismatches as suggested recently by Sapabaev and Laval (11).
EXPERIMENTAL PROCEDURES
Strains and Plasmids—JM253.140 (F- araD139 D(argF-lac)U169
rpsL150 relA1 deoC1 rbsR22 flhD5301 fruA25 mug::mini-Tn10) was
kindly provided by J. Reiss (Princeton University). GM31 (dcm-6 thr-1
hisG4 leuB6 rpsL ara-14 supE44 lacY1 tonA31 tsx-78 galK2 galE2 xyl-5
thi-1 mtl-1) and RP4182 {(flaD-flaP)DE4 trp gal rpsL} are from our
collection. BH156 is GM31 with ung-1 tyrA::Tn10 and was obtained
from M. Lieb (University of Southern California School of Medicine, Los
Angeles, CA). BH161 (BH156 with l DE3 lysogen) was constructed by
A. Beletskii (Wayne State University) using a kit from Novagen (Madison, WI). Construction of BH157 and BH158 is described below. P1vir
phage is from our collection. Plasmid pET11-d was purchased from
Stratagene (La Jolla, CA).
Construction of BH157 and BH158 —BH157 and BH158 were constructed by the P1 transduction of mug::mini-Tn10 from JM253.140
into GM31 and BH156, respectively. LB medium supplemented with
0.2% glucose and 5 mM CaCl2 was inoculated with an overnight culture
of JMR253.140 and was shaken vigorously at 37 °C for 30 min. P1vir
phage was added to the culture at a multiplicity of infection of 0.1, and
the infected cells were further incubated 37 °C for 3 h with continued
shaking. The cells were lysed with chloroform, the culture was centrifuged to clear the cell debris, and the supernatant was removed. The
phage in the supernatant was titered and was used to infect GM31 or
BH156. For the infection, 10 ml of overnight cultures were centrifuged,
1
The abbreviations used are: TDG, thymine-DNA glycosylase; eC,
3,N4-ethenocytosine; Ung, uracil-DNA glycosylase; Mug, mismatched
uracil glycosylase; IPTG, isopropyl-b-D-thiogalactoside; PCR, polymerase chain reaction.
31034
This paper is available on line at http://www.jbc.org
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The human thymine-DNA glycosylase has a sequence
homolog in Escherichia coli that is described to excise
uracils from UzG mismatches (Gallinari, P., and Jiricny,
J. (1996) Nature 383, 735–738) and is named mismatched
uracil glycosylase (Mug). It has also been described to
remove 3,N4-ethenocytosine (eC) from eCzG mismatches
(Saparbaev, M., and Laval, J. (1998) Proc. Natl. Acad.
Sci. U. S. A. 95, 8508 – 8513). We used a mug mutant to
clarify the role of this protein in DNA repair and mutation avoidance. We find that inactivation of mug has no
effect on C to T or 5-methylcytosine to T mutations in E.
coli and that this contrasts with the effect of ung defect
on C to T mutations and of vsr defect on 5-methylcytosine to T mutations. Even under conditions where it is
overproduced in cells, Mug has little effect on the frequency of C to T mutations. Because uracil-DNA glycosylase (Ung) and Vsr are known to repair UzG and TzG
mismatches, respectively, we conclude that Mug does
not repair UzG or TzG mismatches in vivo. A defect in
mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations
due to endogenous damage to DNA in growing E. coli.
Cell-free extracts from mug1 ung cells show very little
ability to remove uracil from DNA, but can excise eC.
The latter activity is missing in extracts from mug cells,
suggesting that Mug may be the only enzyme in E. coli
that can remove this mutagenic adduct. Thus, the principal role of Mug in E. coli may be to help repair damage
to DNA caused by exogenous chemical agents such as
chloroacetaldehyde.
Biological Function of the E. coli Mug
FIG. 1. Excision of uracil with cell-free extracts. The ability of
extracts from ung1 and ung cells to excise uracils from UzG mismatches
was tested. The substrate was a duplex containing the oligos vsr-U and
dcm-94b, and the former oligomer was labeled at the 59 end. The
reaction in which purified Ung was used without UGI served as a
positive control, and the position of the resulting truncated oligomer is
marked with an arrowhead. Following the uracil excision reaction,
DNA was treated with NaOH to cut at the AP site and the products
were separated on a denaturing gel. The total amounts of protein in the
extracts and the presence or absence of UGI in the reaction is noted
above each lane.
within 3 S.D. of the mean, it was included in the set, and the mean and
S.D. of the complete data set were used in further analysis.
Forward Mutation Assays—Appropriate E. coli strains were grown
from single colonies in 5 ml of LB for 24 h at 37 °C with shaking. To
determine the frequency of 5-fluorocytosine-resistant cells, the cultures
were centrifuged to pellet the cells, and the pellets were washed twice
in 5 ml of M63 minimal medium. The cells were ultimately resuspended
in 1 ml of M63 medium and were spread on LB plates to determine the
total number of viable cells. They were also spread on M63 minimal
plates supplemented with 0.1% of casamino acids, 10 mg/ml 5-fluorocytosine, and 20 mg/ml each leucine, threonine, and histidine to determine
the number of 5-fluorocytosine-resistant cells. The plates were incubated for 24 h at 37 °C. The mutant frequency is the number of 5-fluorocytosine resistant cells divided by the total number of viable cells.
To determine frequency of rifampicin-resistant cells, the overnight
cultures were centrifuged to pellet the cells, and the pellets were resuspended in 1 ml of LB. The cells were spread on LB plates or LB plates
containing 100 mg/ml rifampicin. The mutant frequency is the number
of rifampicin-resistant cells divided by the total number of viable cells.
Biochemical Assays for Mug—Five ml of overnight cultures of the
appropriate E. coli strain were used to inoculate 250 ml of LB and
grown at 37 °C to an A550 of 1.0. The cells were harvested by centrifugation, and the pellet was washed with 50 ml of a buffer containing 25
mM HEPES, pH 7.6, and 1 mM ETDA. The cells were again harvested by
centrifugation, and the pellet was resuspended in 20 ml of the lysis
buffer (25 mM HEPES, pH 7.6, 0.5 mM ETDA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Cells were
kept on ice for 30 min and sonicated five times with 30-s pulses.
Between pulses, they were chilled on ice for 30 s. The cell debris was
removed by centrifugation for 30 min at 12,000 rpm 4 °C, and the
supernatant was divided in aliquots. The aliquots were stored at
270 °C.
The DNA oligonucleotides containing uracil (oligo vsr-U) or 3,N4ethenocytosine (eC1) were labeled with 32P at the 59 end and hybridized
to the unlabeled oligomer, oligo dcm-94b, at a molar ratio 1:10 to form
the duplexes with a single U:G or etheno-C:G mismatch. The duplexes
were subjected to treatments with different dilutions of cell-free extracts for 30 min at 37 °C in a treatment buffer (20 mM Tris-HCl, 10 mM
EDTA) and then were treated with 0.1 M NaOH or FA-PY glycosylase to
cleave at the AP sites for another 30 min at 37 °C. The FA-PY glycosylase protein was purified in collaboration with B. Taffe (Wayne State
University). The products were separated in a 20% sequencing gel and
identified by autoradiography and by scanning with a phosphorimager.
The sequences of the oligos used in these experiments were as follows: vsr-U, 59-GACTGGCTGCTACUAGGCGAAGTGCC-39; eC1, 59-
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and the cell pellets were resuspended in 1 ml of a buffer containing 5
mM CaCl2 and 10 mM MgSO4. One hundred ml of the suspensions were
infected with 10, 50, or 100 ml of the P1 phage, and the cultures were
incubated at 37 °C for 30 min without shaking. One hundred ml of 1 M
sodium citrate and 1 ml of LB were added to each tube, and the cells
were incubated for 1 h at 37 °C with shaking. Following concentration
by centrifugation, the cells were spread on LB plates containing 12
mg/ml tetracycline. The plates were incubated overnight at 37 °C, and
three colonies from each transduction were studied further. The DNA
from these colonies was amplified by PCR using the following primers:
primer 1, 59-GATCACCTATCTGCTGGAACAGTACGATCGTG-39; and
primer 2, 59-CTGTATGTCTGCGATGAATCCGGAATG-39. The colonies
that gave rise to a larger PCR product than mug1 cells were chosen for
further analysis.
Confirming the Disruption of mug—The successful transfer of
mug::mini-Tn10 allele was confirmed by Southern hybridization. The
region flanking the mug gene in BH156 chromosomal DNA was amplified by PCR using the following primers 1 and 2 mentioned above. The
2.6-kilobase pair PCR product was excised from a low melting point
agarose gel and labeled with nonradioactive digoxigenin using the
digoxigenin High Prime DNA labeling and detection starter kit II
(Roche Molecular Biochemicals). Chromosomal DNAs from BH156,
BH157, and BH158 were digested with PvuII, and the fragments were
separated in 0.7% agarose gel. The DNA was blotted onto a nylon
membrane and hybridized with probe labeled with digoxigenin. After
treatment with alkaline phosphotase-conjugated antibodies raised
against digoxigenin, the membrane was incubated with solution containing the chemiluminescence substrate for the enzyme CSPD and
exposed to x-ray film for 1 h. The autoradiograph showed that the mug1
strain BH156 contains a 3.0-kilobase pair PvuII fragment containing
mug, whereas the corresponding fragment in BH157 and BH158 is 6
kilobase pairs. This confirms the disruption of mug in BH157 and
BH158.
Construction of an Overproducer of Mug—The open reading frame of
mug1 gene was amplified from chromosomal DNA of RP4182 using the
following primers: primer 3, 59-CCCGCTCTATCGCGGATCAGGCGCGCA-39; and primer 4, 59-CCCCCCCATGGTTGAGGATATTTTGGCTCCAGGG-39. The amplification was done with the Pfu DNA
polymerase, and the ;500-base pair PCR product was isolated from a
low melting agarose gel. The DNA was digested with NcoI and MboI
(compatible with BamHI), and ligated to pET11-d expression vector
digested with NcoI and BamHI. Two clones with the expected inserts
were picked for further analysis. The plasmids were transformed into
BL21(DE3), and one transformant from each plasmid was grown in LB
medium containing 1 mM IPTG. The cells were broken by sonication,
and the cell debris was removed by centrifugation. The supernatant
was subjected to SDS-polyacrylamide gel electrophoresis and stained
with Coomassie Brilliant Blue. Cell-free extract from one of the two
clones showed the presence of a new protein of the expected size. This
plasmid clone, pF168, was used in further studies. To confirm that
pF168 bears a wild-type copy of the mug gene, both strands of the insert
were sequenced using the following primers: primer 5, 59-CTAGTT
ATTGCTCAGCGGTGGCAGC-39; and primer 6, 59-TATAGGGGAATTGTGAGCGGATAAC-39. The sequence of the cloned mug1 was compared with the sequence in the GenBankTM data base (GenBankTM
accession number U28379), and the two sequences were identical.
Genetic Reversion Assays—The genetic reversion assays were performed as described previously (12), except in the case where the cells
carried the plasmid pF168. In this case, E. coli strain (BH161), carrying
the overproducer pF168, was electroporated with pAKS2. The latter
plasmid contains the kan allele cloned in pACYC184 (13). Following
plating, three independent colonies were picked and grown in 10 ml of
LB containing 50 mg/ml carbenicillin and 20 mg/ml chloramphenicol at
37 °C until the A550 reached 0.3. One hundred ml of each culture was
used to inoculate 10 ml of prewarmed LB containing appropriate antibiotics and 30 mM IPTG. The cells were again grown till A550 reached
0.3. The cells were centrifuged at 3000 3 g for 10 min, and the cell pellet
was resuspended in 1 ml of LB. Appropriate dilutions of these cultures
were spread on LB plates to determine the number of viable cells, and
the remaining culture was spread on kanamycin plates to determine
the number of revertants.
The principal source of variation in mutation frequency data is the
existence of mutational “jackpot” (14). We eliminated such data points
from our data sets using the following procedure: the data point suspected of being from a jackpot was set aside and the mean and S.D. of
the remaining data points were calculated. If the suspected data point
was greater than 3 times the S.D. away from the mean, it was declared
to be a jackpot and eliminated from the data set. If the data point was
31035
31036
Biological Function of the E. coli Mug
TABLE I
Repair of UzG and TzG mismatches
Genetic
backgrounda
Revertant frequencyc
Growthb
mug
dcm ung1
Exponential (8)
Stationary (4)
Exponential (8)
Stationary (6)
Exponential (3)
dcm ung
dcm1 ung1 vsr
1
mug
(1.4 6 0.6) 3 1027
(4.2 6 1.2) 3 1027
(1.7 6 0.2) 3 1026
(5.7 6 2.4) 3 1026
(2.1 6 0.8) 3 1026
(1.1 6 0.6) 3 1027
(4.9 6 2.1) 3 1027
(2.6 6 1.3) 3 1026
(7.0 6 2.7) 3 1026
(1.3 6 0.6) 3 1026
The following strains were used in these assays- GM31 (dcm ung1), BH157 (dcm ung1 mug), BH156 (dcm ung), BH158 (dcm ung mug), GM31
with pDCM72 (dcm1 ung1 vsr), and BH157 with pDCM72 (dcm1 ung1 vsr mug).
b
The number of independent cultures used in each experiment is shown in parentheses.
c
Mean 6 S.D.
a
TABLE II
UzG repair in Mug overproducer
Genetic backgrounda
ung mug1
1 (3)
2 (3)
Revertant frequencyb
No IPTG
With IPTG
(3.8 6 2.1) 3 1027
(3.5 6 0.7) 3 1027
(1.4 6 0.9) 3 1027
(4.0 6 2.2) 3 1027
a
Strain BH161. The number of independent cultures used in each
experiment is shown in parentheses.
b
Mean 6 S.D.
FIG. 3. Uracil removal activity in a Mug overproducer. The
excision of uracil by purified Ung and by cell-free extract from BH161
with pF168 are shown. The position of the resulting product is marked
with an arrowhead.
GACTGGCTGCTAC(eC)AGGCGAAGTGCC-39; and dcm-94b, 59-GGCACTTCGCCTGGTAGCAGCCAGTC.
RESULTS
Uracil Excision by Mug—Duplex DNA containing a UzG mismatch was treated with cell-free extract prepared from ung
mug1 cells. The DNA was further treated with NaOH to convert the abasic sites created by the extract to strand breaks,
and the products were separated on a denaturing gel. The
reactions were carried out in pairs, one with the Ung inhibitor
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FIG. 2. Induction of Mug from a T7 promoter. SDS-polyacrylamide gel electrophoresis gel of extracts prepared from BH161 cells
containing a plasmid are shown. The amount of IPTG used to induce the
T7 promoter is shown above each lane. Lanes 1 and 2, pET11-d; lanes
3– 8, pF168. The position of the induced protein is marked with an
arrowhead.
UGI (15) and the other without the inhibitor. The use of UGI in
one reaction assures that any uracil excision activity seen in
that reaction must be due to an enzyme other than Ung.
The cell extract contained a weak uracil glycosylase activity
(Fig. 1A, lane 4) that was resistant to UGI (lane 5). This activity
was reproducible and is probably the same activity reported by
Gallinari and Jiricny (10). Compared with this weak activity,
the uracil excision activity in extracts prepared from ung1
mug1 cells was much easier to detect. Whereas 8 mg of cell-free
extract from ung cells had barely detectable activity, 38 ng of
extract from ung1 cells cleaved all the uracil in DNA (Fig. 1B,
lane 7). The latter activity was completely inhibited by UGI
confirming that it was due to Ung. Based on these and other
results we conclude that uracil excision activity in E. coli due to
Ung is at least 400 times greater than uracil excision due to
any other enzyme including Mug.
Role of Mug in the Repair of UzG or TzG Mismatches—We
used a genetic reversion system involving a defective kanamycin resistance gene (13) to assess the role of Mug in avoiding
spontaneous C to T mutations. The reversion to kanamycin
resistance results exclusively from C to T mutations at a site
for cytosine methylation and either a 5-methylcytosine to T
change or C to U to T change can be studied with this system
in appropriate genetic backgrounds. TzG and UzG mismatches
are, respectively, the intermediates in these mutagenic pathways, and hence any excision of T or U by Mug should reduce
C to T mutations.
We compared the antimutagenic effects of Mug to those of
Ung and Vsr using this assay. The presence of Mug did not
affect mutations by either pathway (Table I). Whereas the
presence of Ung reduced the reversion frequency by a factor of
;10, Mug did not significantly affect the frequency. We have
previously shown that in a mug1 strain, Vsr reduces the frequency of 5-methylcytosine to T mutations by a factor of about
4 (16). In contrast, Mug reduced these mutations only slightly,
and this reduction was not statistically significant (Table I).
In the experiments discussed above, cells were dividing at
the time of their selection for kanamycin resistance. To assess
the role of Mug in stationary phase of cell growth, cells were
shaken at 37 °C for ;24 h and then plated to select for kanamycin-resistant revertants. Growing the cells to stationary
phase increased the reversion frequency by a factor of ;2
compared with growing cells, but there was no significant effect
of Mug on the mutant frequencies (Table I). In contrast, the
Biological Function of the E. coli Mug
31037
TABLE III
Forward mutation frequencies
Mutant frequencya
Genetic
background
ung (3)c
Rifampicin resistance
5-Fluorocytosine resistance
mug1b
mugb
mug1
mug
(2.4 6 1.0) 3 1027
(6.5 6 2.9) 3 1027
(4.4 6 1.3) 3 1024
(3.4 6 1.8) 3 1024
Mean 6 S.D.
Strains BH156 (mug1) and BH158 (mug).
c
The number of independent cultures used in each experiment is shown in parentheses.
a
b
Ung defect again increased the mutant frequency by a factor of
approximately 10. Based on these results, we conclude that
Mug does not play a significant role in the repair of UzG or TzG
mismatches in E. coli.
Effects of Overexpression of Mug on UzG Repair—It seemed
possible that the inability of mug1 ung cells to repair UzG
mismatches was due to inadequate expression of Mug in the
cells. To see whether overexpression of Mug in the cells can
reduce C to T mutations, the mug1 gene was cloned in a
multicopy plasmid and expressed from a bacteriophage T7 promoter. A plasmid carrying this construct was introduced into
mug1 ung cells along with the tester plasmid containing the
kan gene. The expression of Mug was optimized by varying the
concentration of IPTG used to induce the promoter, and the
level of Mug in the cells was monitored by gel electrophoresis.
The amount of free Mug in the cells increased with IPTG
concentration, reaching a maximum between 20 and 100 mM of
the inducer (Fig. 2). The level of soluble Mug did not increase at
higher concentrations of the inducer, probably because Mug
tends to aggregate at high concentrations.2 The cell viability is
also low at concentrations above 40 mM. Consequently, the
assays were done at 30 mM IPTG.
Cell extracts containing high levels of Mug are able to excise
uracils from DNA (Fig. 3, lane 4). In this case, nearly all the
uracil was excised from UzG mismatches regardless of the
2
E. Lutsenko and A. S. Bhagwat, unpublished results.
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FIG. 4. Ethenocytosine removal activity in cell-free extracts.
The ability of extracts from mug1 and mug cells to excise ethenocytosine from eCzG pairs was tested. The substrate was a duplex containing the oligos eC1 and dcm-94b, and the former oligomer was labeled at
the 59 end. The reaction conditions were similar to those described in
legend to Fig. 1, except that the FA-PY glycosylase protein was used to
cleave the abasic site. The expected position of the final reaction product was identified by excising uracil from UzG pairs using Ung and
electrophoresing the reaction products in an adjacent lane (not shown).
The expected position of the product is marked with an arrowhead.
presence of UGI in the reaction (Fig. 3, lane 5). Purified Mug is
also able to excise uracil from UzG mismatches (not shown).
Surprisingly, induction of mug1 had little effect on the frequency of C to T mutations. In one data set there was a slight
decrease in mutant frequency as a result of Mug overproduction, but this effect was not reproducible (Table II). The mutant
frequencies reported in this table are lower than that in Table
I, because in this case the kan gene was on a low copy number
plasmid. Induction of the T7 promoter with lower concentrations of IPTG or with 40 mM IPTG also did not affect the mutant
frequency (not shown), suggesting that even at high concentrations, Mug cannot effectively substitute Ung to repair UzG
mismatches in DNA.
Effect of Mug on Forward Mutations—We wondered whether
Mug could repair endogenous DNA damage other than UzG and
TzG mismatches. If this were true, a mug mutation would have
a mutator phenotype. We tested this possibility by comparing
the frequencies of rifampicin-resistant and 5-fluorocytosineresistant mutants in mug1 and mug strains.
The results were largely negative (Table III). The frequency
of rifampicin-resistant mutants was slightly higher in a mug
strain, but the the frequency of 5-fluorocytosine-resistant mutants was the same in the two genetic backgrounds. It is possible that Mug does prevent a small number of mutations and
that this is not evident in the 5-fluorocytosine-resistant mutation assay because the background frequency of mutations is
high in this assay (Table III). In any case, mug is at best a very
weak mutator and hence is unlikely to be important for correcting endogenous damage to DNA in E. coli.
3,N4-Ethenocytosine Excision by Mug—A wide range of
chemicals react with bases in nucleic acids to form ethenobases. These chemicals include vinyl compounds, haloaldehydes, a-haloketones, haloalkanes, halothioketenes, haloketenes, and products of lipid peroxidation (17, 18). After the
work presented above was completed, Saparbaev and Laval
purified a protein from E. coli that removes 3,N4-ethenocytosine (eC) from DNA and showed that it was Mug (11). Purified Mug excised eC from duplex DNA with eCzG pairs, but not
from single-stranded DNA. It also excised uracils, but its catalytic efficiency for the removal of eC excision was 50 times
higher than for the removal of uracil from UzG mismatches
(11).
We have confirmed the ability of Mug to excise eC from DNA.
Cell-free extracts from cells containing overproduction of Mug
and purified Mug excised eC from DNA (not shown). We also
wanted to find out whether eC removal activities other than
Mug existed in E. coli. For this purpose, we used the mug strain
described above.
When duplex DNA containing a eCzG pair was treated with
various cell-free extracts in a manner similar to that described
for UzG mispairs, removal of eC was readily detected in mug1
extracts (Fig. 4). This treatment converted the labeled substrate to shorter products of expected length or products that
were ;1 nucleotide longer or shorter (Fig. 4, lane 2). Although
the shorter product is likely to have resulted from the action of
an AP endonuclease in the extract to the 59 side of the abasic
31038
Biological Function of the E. coli Mug
site created by Mug, the source of the longer product is not
known. Regardless, these results confirm the existence of eC
removal activity in E. coli (11). Furthermore, extract prepared
from mug cells did not possess the ability to create abasic sites
at the eC (Fig. 4, lane 3). Thus mug appears to code for the
principal eC excision activity in E. coli.
DISCUSSION
Acknowledgments—We are grateful to J. Reiss (Princeton University) for providing a bacterial strain and to A. Beletskii (Wayne State
University) for constructing a phage lambda lysogen.
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We have shown here that although concentrated cell extracts
containing Mug can be used to show a small amount of excision
of uracils from DNA, this enzyme does not act as a UzG correction enzyme in growing or stationary E. coli. Furthermore, Mug
does not appear to play a significant role in repairing any other
DNA damage that occurs spontaneously in the cells. Mug is
clearly more efficient at excising eC from eCzG pairs, and this
may be the only activity of the kind in E. coli.
It is surprising that despite its ability to excise uracils from
UzG mispairs, overproduction of Mug from a strong T7 promoter does not result in the reduction of C to T mutations
(Table II). A possible reason for this apparent inactivity of Mug
in vivo is that Mug may aggregate to form inclusion bodies.
Consistent with this hypothesis we have found that purified
Mug rapidly aggregates to form stable high molecular weight
complexes. As a result, very little Mug may be available to
repair the mismatches despite overproduction.
An alternate possibility is that C to U deaminations mostly
occur in single-stranded regions of the genome and that Mug is
unable to excise these uracils because of its strict requirement
for UzG mismatches. In contrast, Ung acts preferentially on
uracils in single-stranded DNA (19, 20) and should efficiently
repair such uracils. If so, Mug is poorly suited to be a backup
enzyme for Ung.
At this time, the biological role of Mug in E. coli remains a
matter of speculation. If the role is in the removal of eC from
DNA—and this is very likely—then the lack of a strong mutator phenotype for mug is not surprising. Defects in genes that
code for enzymes that repair alkylated bases also do not have a
mutator phenotype. This has been interpreted to mean that
there is little alkylation damage to DNA bases in exponentially
growing E. coli. By analogy, it is likely that there is very little
endogenous eC, or any other damaged base that may be removed by Mug, in E. coli.
It is important to note that some of the enzymes involved in
the repair of DNA damage caused by exogeneous agents are
induced by the damaging treatment. These include the the
nucleotide excision proteins and the 3-methyl adenine glycosylase II, AlkA. It would be interesting to know whether mug1 is
similarly inducible in response to damage to cellular DNA.