Multiple Mutant of Escherichia coli Synthesizing Virtually
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JOURNAL OF BACTERIOLOGY, July 1992, p. 4450-4456 Vol. 174, No. 13
0021-9193/92/134450-07$02.00/0
Copyright X) 1992, American Society for Microbiology
Multiple Mutant of Escherichia coli Synthesizing Virtually
Thymineless DNA during Limited Growth
HIYAM H. EL-HAJJ,t LINGHUA WANG, AND BERNARD WEISS*
Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602
Received 13 December 1991/Accepted 21 April 1992
The dut gene of Escherichia coli encodes deoxyuridine triphosphatase, an enzyme that prevents the
incorporation of dUTP into DNA and that is needed in the de novo biosynthesis of thymidylate. We produced
a conditionally lethal dut(Ts) mutation and isolated a phenotypic revertant that had a mutation in an unknown
gene tentatively designated dus (for dut suppressor). The dus mutation restored the ability of the dut mutant to
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grow at 42°C without restoring its enzymatic activity or thymidylate independence. A strain was constructed
bearing, in addition to these mutations, ones affecting the following genes and their corresponding products:
ung, which produces uracil-DNA N-glycosylase, a repair enzyme that removes uracil from DNA; deoA, which
produces thymidine (deoxyuridine) phosphorylase, which would degrade exogenous deoxyuridine; and thyA,
which produces thymidylate synthase. When grown at 42°C in minimal medium containing deoxyuridine, the
multiple mutant displayed a 93 to 96% substitution of uracil for thymine in new DNA. Growth stopped after
the cellular DNA had increased 1.6- to 1.9-fold and the cell mass had increased 1.7- to 2.7-fold, suggesting a
general failure of macromolecular biosynthesis. DNA hybridization confirmed that the uracil-containing DNA
was chromosomal and that new rounds of initiation must have occurred during its synthesis.
The DNA of almost all organisms contains thymine rather replace a chromosomal dut+ gene unless there were another
than uracil. A plausible reason was provided by Lindahl (22). functional copy of dut within the cell. We postulated at first
Uracil can arise in DNA from the mutagenic spontaneous that the lethality might be the result of excessive incorpora-
hydrolysis of DNA cytosine, and it is recognized and re- tion of uracil into DNA; the uracil-containing chromosome
moved by a ubiquitous repair enzyme, uracil-DNA N-glyco- might function poorly or be irreparably broken during at-
sylase. Therefore, thymine, rather than uracil, was estab- tempted excision repair (10, 11). However, we could not
lished as a normal constituent of DNA. However, phage restore viability by supplying large amounts of exogenous
PBS2 of Bacillus subtilis possesses uracil-containing DNA thymidine or by producing combinations of mutations that
(11), indicating that at least under some circumstances, would reduce the formation of dUTP or the removal of uracil
thymineless DNA can function normally. To what extent from DNA. It is possible, therefore, that the lethality of the
have other organisms become dependent on thymine-con- dut mutation might be unrelated to uracil incorporation into
taining DNA, and what other properties, if any, are unique DNA. To study the mechanism of killing, we have in this
to such DNA? To answer these questions, we have isolated study isolated a conditionally lethal dut mutant as well as a
mutants of Escherichia coli that incorporate uracil into DNA new mutation in a gene called dus, which restores the
in place of thymine. viability of dut mutants without restoring their dUTPase
In E. coli, most of the thymidylate needed for DNA activity. We also report some preliminary experiments in
synthesis is manufactured via dUTP with the help of deoxy- which a strain of E. coli that contains these mutations in
uridine triphosphatase (dUTPase) (27, 33). dUTPase cata- combination with others was able to synthesize virtually
lyzes the hydrolysis of dUTP to PPi and dUMP, a substrate thymineless DNA for almost one generation.
for thymidylate synthase. Therefore, mutations in dut, the
gene for dUTPase, cause an increased level of dUTP and a
corresponding decrease in dTTP so that large amounts of MATERIALS AND METHODS
uracil are incorporated into DNA in place of thymine. This
incorporation is only transient (35) because uracil is removed Microbial strains and plasmids. The bacterial strains used
from DNA via an excision repair initiated by uracil-DNA (Table 1) were derivatives of E. coli K-12. The recombinant
N-glycosylase, the product of the ung gene (11). In dut ung plasmids pIT15 (34) and pLW2 are derivatives of pBR322
double mutants, the misincorporated uracil is not effectively (31) that were arbitrarily chosen as hybridization probes for
excised; in one study, there was a stable replacement of up chromosomal DNA. pIT15 bears the soxRS region of E. coli
to 19% of DNA thymine by uracil (37). We wished to obtain on a 2.5-kb insert. Plasmid pLW2 bears the dcd gene (36) on
higher levels of replacement, but we were hampered by the a 1.3-kb HindIII-EcoRV fragment of E. coli DNA subcloned
leakiness of the existing dut mutations. Therefore, in a from X2E1 (miniset no. 355) from the library of Kohara et al.
previous study, we generated a tight dut mutation by insert- (20). Phage XBW112 (dut+) is a c+Q+S+ derivative of
ing a chloramphenicol resistance gene within a plasmid dut XBW111 (32) that was constructed by crossing XBW111 with
gene (14). However, the insertion was lethal; it could not
A wild type. Plasmid pWB30 contains the dut gene on an
881-nucleotide fragment cloned into the EcoRV site of
pBR322. The cloned segment extends from an EcoRV site
*
Corresponding author. 292 nucleotides from the 3' end of dfp to the XmnI site at
t Present address: Department of Medicine, Beth Israel Medical nucleotide 44 of the ttk gene (14, 23). It was obtained from
Center, New York, NY 10003. plasmid pWB2, which is similar to pWB1 (32) except that the
4450VOL. 174, 1992 E. COLI MUTANT SYNTHESIZING THYMINELESS DNA 4451
TABLE 1. Bacterial strains
Strain Description Source or referencea
AT2243-llc HfrC (PO-2A), metBi pyrE41 uhp-1 rel-I tonA22 (X) 15
BD2008 KL16 ung-151::TnlO B. K. Duncanb
BW285 KL16 dut-1 9
BW310 KL16 ung-l This labc
BW322 KL16 pyrE zia-207::TnlO 32
BW386 KL16 recA56 srlC300::TnlO This labc
BW712 AT2243-11c mutL::TnlO P1(D6432) x AT2243-11C
BW719 KL16 ttk-l::kan 14
BW741 KL16 dut-22(Ts) ttk-l::kan P1(BW928) x KL16
BW743 KL16 dut-22(Ts) P1(BW928) x BW322
BW928 BW712 dut-22(Ts) ttk-l ::kan This study
BW929 KL16 dut-22(Ts) ttk-l::kan ung-J P1(BW741) x BW310
BW930 KL16 dut-22(Ts) ttk-1::kan ung-J recAS6 srlC300::TnlO P1(BW386) x BW929
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BW931 KL16 dus-1 dut-22(Ts) ttk-l::kan ung-1 srlC300::TnJO This study
BW933 KL16 deoA22 thr-34::TnlO P1(HH17) x BW35
BW934 KL16 deoA22 P1(KL16) x BW933
BW935 KL16 dut-22(Ts) ttk-l::kan deoA22 P1(BW741) x BW934
BW936 BW935 ung-1S1::TnJO P1(BD2008) x BW935
BW938 KL16 dus-I dut-22(Ts) ttk-l::kan ung-J BW931 -- Srl+d
BW939 BW938 deoA22 thr-34::TnlO P1(BW933) x BW938
BW940 KL16 dus-1 dut-22(Ts) ttk-l::kan ung-J deoA22 BW939 Thr+d
BW942 Same as that for BW940 but ung-151::TnIO P1(BD2008) x BW940
BW943 KL16 dut-22(Ts) ttk-l::kan ung-151::TnlO deoA22 thyA BW942 -*
trimethoprim resistance (26)
BW945 KL16 dut-21::cat ung-J dus-I srlC300::TnlO P1(HH1) x BW931
D6432 mutL::TnlO argE metB gyrA rpoB A(lac-pro) supE? X-F- 16
HH1 KL16 dut-21::cat (XpyrE+ dut+ c1857) 14
HH17 Hfr Reeves 4 (PO100) deoA22 thr-34::TnlO upp-J udp-1 metBl argF58 reL4lA- 14
KL16 Hfr P045 thi-I relAI spoTI 2
a Phage P1 transductions are described as follows: Pl(donor) x recipient.
' Same as that for BD2007 (12), but cured of X.
c Pedigrees available on request.
d Selected for spontaneous precise excision of Tn1O.
cloned DNA is in the opposite orientation. Plasmid pES19 completeness of digestion and separation of mononucle-
(dfp+ dut-19::TnlOOO) was previously described (30). otides from other radiochemical material. Spots produced by
Microbiological methods. Microbiological methods, selec- carrier deoxynucleotides (0.2 ,umol each) were visualized at
tive media, and testing of ung and deoA genotypes were 254 nm and scraped into vials for liquid scintillation count-
described previously (14, 38). For the growth of dut mutants, ing.
even rich media were routinely supplemented with thymi- Filter hybridization of uracil-containing DNA (see Table 4).
dine at 0.5 mM (14). Minimal media were also supplemented Strain BW943 was grown with aeration at 37°C in a low-
with thiamine at 1 ,ugIml. Low-phosphate medium was a phosphate labeling medium (4) containing 0.25 mM
modification of the 32p labeling medium of Bochner and [6-3H]thymidine (25 ,uCi/ml). When the cells reached a
Ames (4), containing 0.5% Norit-treated vitamin-free density of 2.5 x 108 ml-', they were washed twice by
Casamino Acids in place of potassium phosphate. centrifugation in unsupplemented medium and resuspended
Production of the dut-22(Ts) mutation. P1 phages were at 1.25 x 108 ml-l in medium containing 32p; (14 ,Ci/ml) and
grown on strain BW719 (ttk-1::kan). The phage lysate was 0.5 mM deoxyuridine for further growth at 42°C. After 3 h,
treated with 0.4 M hydroxylamine (19) for 18 h at 37°C and the DNA was isolated from 30 ml of culture, denatured by
then used to transduce strain BW712 to kanamycin resis- heating at 100°C for 5 min, and dissolved in 5 ml of
tance at 30°C in the presence of thymidine. hybridization fluid. Molecular hybridization was performed
DNA isolation. E. coli DNA was extracted from cells and (28) with 2 ,ug each of the indicated target DNAs bound to
purified as described previously (39). Plasmids were ampli- nitrocellulose filters.
fied with chloramphenicol and purified by a rapid alkaline Other methods. dUTPase assays (30) were performed on
extraction method (28). Residual RNA was removed from all sonicates of growing cells (9). Protein was determined by the
DNA preparations by digestion with RNase A, and the DNA bicinchoninic acid method (29). Relative cell mass and cell
was precipitated with ethanol (28). concentration were determined by turbidity (6). The concen-
DNA composition. Purified cellular DNA was digested to tration of cellular DNA was measured with diphenylamine
mononucleotides by pancreatic DNase and venom phospho- (5), and that of purified plasmid DNA was estimated by
diesterase (7). The mononucleotides were separated by staining with ethidium bromide (28).
two-dimensional thin-layer chromatography on polyethyl-
eneimine cellulose (4). The solvent for the first dimension RESULTS
was 1 M acetic acid adjusted to pH 3.5 with NH40H; that for
the second dimension was isobutyric acid-concentrated Isolation of a dut(Ts) mutant. To study the lethal effects of
NH40H-0.5 M potassium phosphate buffer (pH 3.5)-H20 dut mutations and to produce other mutations that would
(66:1:2:31). Autoradiograms were developed to confirm restore viability, we first had to isolate a dut mutant that we4452 EL-HAJJ ET AL. J. BACTERIOL.
could propagate, i.e., a conditionally inviable strain. The
technique of Hong and Ames (19) was used to generate
mutations within 2 min of the ttk-1::kan allele. ttk is the
second member of the two-gene dut operon. It encodes a
23-kDa polypeptide of unknown function, and its mutants 4
have no discernible phenotype (14). The ttk-l::kan insertion
mutation (14) consists of a kanamycin resistance determi-
nant of Tn9O3, which was ligated into a site at nucleotide 258
of the 633-bp open reading frame of ttk (14). A phage P1
lysate of a ttk-l: :kan mutant was treated with hydroxylamine
and used to transduce another strain to kanamycin resis-
tance at 30°C as described in Materials and Methods. Of
26,000 transductants that were screened by replica plating, 0
110 had a temperature-sensitive kanamycin resistance.
Twenty others were conditional lethal (Ts) mutants, and of 0)
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these, 14 were complemented by XBW112 and therefore had
mutations in a 7-kb region spanning the dut gene. Seven of
these 14 mutants hadVOL. 174, 1992 E. COLI MUTANT SYNTHESIZING THYMINELESS DNA 4453
TABLE 2. dUTPase activities of strains bearing dut and A
dus mutations dCMPQ C
Grwh
Grwh dUTPase
Temp
iD l dTMP
Strain Relevant genotype temp
(OC)
(relative sp act)a coefficient
(Q430b)
300C 42°C dUMPA OdTMP dU
rUMP
J
KL16 Wild type 30 (1.0) 1.6 1.6
BW285
BW741
BW931
dut-1
dut-22(Ts)
dut-22(Ts) dus-1
30
30
30
0.013
0.33
0.40
0.0088
0.57
0.65
-c
1.7
1.6 X-
..
dGMP
j: 6
BW945 dut-21::cat dus-1 30 0.0052 0.0054
KL16
BW931
Wild type
dut-22(Ts) dus-l
42
42
1.8
-
2.8
0.20
1.6 Pj3)fV
(D i
a Enzymatic activity is reported as a ratio of specific activity of a mutant
strain to that of a wild-type (dut+ dus+) strain grown and assayed at 300C. A FIG. 2. Two-dimensional thin-layer chromatography of 32P-la-
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relative specific activity of 1.0 corresponds to 5.54 U of dUTPase per mg of beled DNA nucleotides isolated from a dut(Ts) ung dus deoA thyA
protein. mutant. Strain BW943 was grown to a density of 2.5 x 108 ml-' at
b Q30 is the ratio of dUTPase activity at 42°C to that at 30°C. 37°C in a thymidine-supplemented tryptone-yeast medium, washed
c, not calculated or not done. by centrifugation, and transferred to low-phosphate medium at 42°C
containing 32Pi at 10 ,Ci ml-' and either 0.5 mM deoxyuridine or 0.5
mM thymidine. The cells were harvested at 2 h, by which time
growth had ceased. DNA was extracted and digested to mononu-
Tn9 that specifies chloramphenicol resistance into the mid- cleotides that were separated by chromatography (see Materials and
dle of the dut gene. Previously, a dut-21::cat would only be Methods). (A) The positions of markers visualized by UV or
tolerated in merodiploids that had a functional second copy autoradiography are shown. Spot X, visible only on autoradiograms
of dut (14). Now, we could transduce it into a dus-1 mutant (panels B and C), probably represents undigested oligonucleotides.
rUMP (dashed outline) was not visible on the autoradiograms
that was haploid for dut, producing the strain BW945 (Table (panels B and C). (B) Cells were grown with deoxyuridine. Positions
1). The transduced chloramphenicol resistance was geneti- of UV-visible dTMP and dUMP markers are indicated. (C) Cells
cally stable, indicating that we had indeed replaced dut- were grown with thymidine.
22(Ts) and had not merely inserted dut-21::cat into a tan-
demly duplicated dut region. The suppression of the
transduced dut-21: :cat allele confirmed that dus-1 is an
extragenic mutation and suggested that it is not a transla- mutation should prevent the synthesis of dTMP from the
tional suppressor. dUMP that might arise either through residual dUTPase
dUTPase levels. Although the dus-1 mutation suppressed activity or from another pathway (e.g., the hydrolysis of
the lethality of dut-22(Ts) and dut-21::cat mutations, it did dUDP). The deoA (thymidine [deoxyuridine] phosphorylase)
not affect dUTPase levels (Table 2). The properties of the mutation should enable the more efficient utilization of
mutant dUTPase specified by the dut-22(Ts) allele were exogenous thymidine or deoxyuridine (14).
contrary to our expectations (Table 2). First, its residual In the experiment whose results are shown in Fig. 2, the
activity at 42°C was higher than that of a dut-i mutant that is multiple mutant was grown for 2 h at 42°C in a medium
not temperature sensitive for growth (18). Therefore, either containing 32p; and either thymidine or deoxyuridine. The
the relative dUTPase activities of the mutant enzymes in cells that were fed deoxyuridine appeared to have almost
vitro do not reflect their activities in vivo or conditional completely replaced dTMP by dUMP in newly synthesized
lethality is related to some function of the mutant protein DNA (Fig. 2B); radioactivity measurements indicated a 91%
other than its dUTPase activity. Second, the dut-22 enzyme replacement. In contrast, the DNA synthesized in the pres-
did not have an altered temperature coefficient. Third, in a ence of thymidine (Fig. 2C) contained dUMP amounting to
separate experiment, we found that in crude extracts, the no more than 3% of the dTMP. No radioactivity was seen in
mutant and wild type had similar Km values at 42°C (6 and 7 the rUMP (ribouridylate) spots, indicating the absence of
puM, respectively). One finding, however, indicated the significant contamination of the DNA samples by RNA.
probable basis for the thermosensitive phenotype. When the The experiment whose results are shown in Fig. 2 was
dut(Ts) mutant was grown at 30°C, it had 36% of the repeated with monitoring of growth and of DNA synthesis.
dUTPase activity of the wild type, but when grown at 42°C, When we attempted to measure rates of DNA synthesis by
it had only 7% (Table 2). Therefore, the mutant enzyme (or the uptake of radioactive thymidine, the results immediately
its production) appears to be heat labile in vivo. after the medium shift were erratic, suggesting a fluctuation
Incorporation of uracil into DNA. The availability of viable of nucleotide pools. Therefore, we chemically measured the
strains containing tight dut mutations enabled us to try to DNA content of the cells. In the experiments whose results
replace all of the thymine in DNA with uracil. We con- are shown in Table 3, a growing culture of the multiple
structed BW943, a strain with the following relevant geno- mutant was shifted from a complex thymidine-supplemented
type: dut-22(Ts) dus-i ung-Si ::TniO thyA deoA. The strain medium to a thymidine-free medium containing a high con-
was viable at 42°C. The rationale for its construction was as centration of deoxyuridine. The culture was then incubated
follows. The dut mutation should lead to the accumulation of at 42°C for 2 h, by which time it reached an apparent limit of
dUTP and to its incorporation into DNA at a high tempera- growth that was less than 25% of that displayed by wild-type
ture. dus-i, which suppresses the lethality of dut, would cells in a separate experiment. During this time, the cell
favor continued growth of the cells under nonpermissive mass of the culture increased about 1.7- to 2.7-fold and its
conditions. The ung mutation would block the removal of DNA content almost doubled. In the new DNA, uracil
uracil from the DNA, and the ung-iSi ::TniO insertion might residues replaced 93 to 96% of the thymine.
be tighter than ung-1. The thyA (thymidylate synthase) In the experiment whose results are shown in Table 3,4454 EL-HAJJ ET AL. J. BACTE RIOL.
TABLE 3. Synthesis and composition of DNA in a dut(Ts) ung TABLE 4. Identification of uracil-containing DNA by
dus deoA thyA mutant grown at 42°C in the presence filter hybridizationa
of deoxyuridinea
Unlabeled Uracil-DNA Genomic DNA Ratio
Changes over 2 h target
DNAb
bound
(32p cpm)
bound
(3 H cpm) (
R32poH)
Expt Increase in Increase Uracil content of
cell mass in DNA new DNA [U/(U+T)I E. coli 3,578 2,403 1.5
(fold) (fold) (%) pLW2 955 620 1.5
pIT15 431 265 1.6
1 1.7 1.8 93 pBR322 5 11
2 2.7 1.9 96
a Strain BW943 was grown first at 37°C in a medium containing [3H]thymi-
a Strain BW943 was grown at 37°C to 1 x 108 ml- (experiment 1) or 2.5 x
dine and then at 42°C in a medium containing 32Pi and deoxyuridine. The DNA
108 ml-' (experiment 2) in a thymidine-supplemented tryptone-yeast medium, was extracted and hybridized with the indicated target DNAs (see Materials
washed, and transferred at time zero to a 32p labeling medium containing and Methods). Values for a blank filter (46 cpm of 32p; 20 cpm of 3H) were
deoxyuridine for growth at 42'C. The experimental conditions were the same subtracted from the results.
as those described in the legend to Fig. 2. b Plasmids pLW2 and pIT15 are derivatives of pBR322 containing segments
of E. coli DNA from regions at 45 and 92 min, respectively, of the chromo-
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somal linkage map (3). (See Materials and Methods.)
growth stopped 1.5 h after the shift to deoxyuridine-contain- DISCUSSION
ing medium, and there was no further increase even after an
additional 18 h of incubation. In a control experiment, The vital nature of the dut gene was previously demon-
cultures were grown at 42°C in minimal medium supple- strated via the lethality of a dut insertion mutation (14), but
mented with thymidine rather than deoxyuridine. The cells the mechanism of cell death is unknown. The lethality was
grew continuously to saturation. not reversed by mutations in known genes affecting dUTP
We do not know at this point what effect a dus+ allele formation or the fate of uracil-containing DNA. Therefore,
would have had on the outcome of the experiments whose we were led to obtain a conditionally lethal dut mutant and to
results are shown in Table 3. However, the presence of the use it to select for extragenic mutations that would suppress
the lethality. Our dut(Ts) mutant was inviable at 42°C, had a
dus-1 mutation enabled us to conclude that the cessation of low level of dUTPase at that temperature, and was comple-
cell growth in the presence of deoxyuridine at 42°C could not mented by a dut+ plasmid. A mutation in an unknown gene,
be attributed to the conditional lethality of dut-22 per se. dus, suppressed the lethality without restoring dUTPase
Moreover, the dus-1 mutation permitted us to repeat this activity. We reasoned that by ultimately identifying the dus
experiment with an analog of the multiple mutant containing gene product and by studying nucleotide pools in dut(Ts) and
a tighter dut mutation, dut-21::cat. The results were similar. dus mutants, we might learn why the dut gene is vital. We
Cell growth stopped by 2 h, by which time cell mass had also hoped that by suppressing the lethality of dut without
increased 2.5-fold. The ratio of uracil to uracil plus thymine restoring dUTPase, we might maintain cells with high levels
in the total cellular DNA was measured by high-performance of uracil in their DNA. This study contains our first such
liquid chromatography (21) and equaled 46%, a result that is attempt. We were able to synthesize new chromosomal
compatible with the synthesis of almost a full strand of DNA in which over 90% of the thymine was replaced by
uracil-containing DNA. uracil before there was a shutdown of DNA synthesis and
The uracil-containing DNA is chromosomal. The experi- cell growth.
ments described above entailed thymine deprivation, a con- Our results suggested that dus-1 is not a translational
dition that can induce cryptic prophages (8, 25). Therefore, suppressor and that the suppressor mutations occur with
we used DNA-DNA hybridization to identify the newly such high frequency (10-5) that they might be null muta-
synthesized DNA. The conditions were similar to those tions. These assumptions have been confirmed recently by
described in Table 3, footnote a, except that the cells were the isolation of additional suppressor mutations by transpo-
labeled with [3H]thymidine during preliminary growth in son insertion and the demonstration that they belong to the
thymidine-enriched medium and with 32Pj during subsequent same complementation group that dus-1 does. In work to be
growth with deoxyuridine. During growth in deoxyuridine, published separately, Wang and Weiss (36) present evidence
that dus-1 is actually a dcd allele; it is a mutation in the
the DNA content of the culture increased 1.6-fold. The DNA structural gene for dCTP deaminase, the enzyme that pro-
was isolated and tested for its ability to hybridize with whole duces about 75% of the dUTP in E. coli (27). This finding
genomic DNA and with cloned segments of chromosomal suggested that it is the accumulation of dUTP that causes the
DNA. The 3H label served as a convenient measurement of lethality associated with dut mutations.
the relative concentration of cellular DNA and of the overall In our dut mutants, dUTPase activity did not seem to be
hybridization efficiency, whereas the 32P radioactivity was a correlated with viability. At 42°C, the dUTPase of a viable
specific tracer for the newly synthesized uracil-containing dut-I mutant had 0.6% of the activity of the wild-type
DNA. If the new 32P-labeled DNA were that of an induced enzyme, whereas the temperature-sensitive dut-22 mutant
prophage, it should not hybridize to two plasmids carrying had at least 7% residual activity. Superficially, the results
widely separated chromosomal segments. However, the suggest that lethality is not associated with dUTPase activity
newly synthesized 32P-DNA and genomic 3H-DNA annealed but with some other undiscovered activity of the enzyme.
in similar ratios to whole genomic DNA and to the recom- However, because dCTP deaminase mutations suppress dut
binant plasmids (Table 4). A vector DNA (pBR322) control lethality, it is likely that viability is directly related to
demonstrated the specificity of the hybridization. The results dUTPase levels. Therefore, our measurements of dUTPase
indicate that the newly synthesized uracil-containing DNA is activity in crude extracts may not reflect those in the living
chromosomal and is not that of an induced prophage. cell. Given the important role of the enzyme, it is notVOL. 174, 1992 E. COLI MUTANT SYNTHESIZING THYMINELESS DNA 4455
surprising that a mutant with only 7% residual activity is topoisomerases, or transcriptional regulators to recognize
inviable. What is harder to explain is how a dut-I mutant, uracil-containing DNA. However, if there are critical thy-
which appears to have less than 1% residual activity, is mine residues in vital DNA-protein recognition sites, there
viable. We should like to suggest that our measurements of probably cannot be many such sites because E. coli main-
dUTPase activity in our dut-I mutant may have been falsely tains vigorous growth in the face of at least 10% replacement
low because of inactivation of the mutant enzyme during the of thymine by uracil (11). There may even be a selective
preparation of cell extracts. A similar effect has been noted evolutionary pressure against the requirement for thymine at
with mutants for other vital enzymes, e.g., valine- and important sites because transient incorporation of dUTP,
phenylalanine-tRNA ligases; extracts prepared from temper- which occurs in wild-type cells, might interfere with the
ature-sensitive conditional lethal mutants had 0.3% residual function of those sites. Consequently, it might be possible to
activities even at a permissive temperature (13). select for additional bacterial mutations or to find phages and
During the extensive incorporation of uracil into DNA, the plasmids that will allow the extensive synthesis of thymine-
DNA content of the cells nearly doubled (Table 3). There- less DNA in our mutants, thereby further enabling us to
fore, the cellular DNA should be expected to consist almost explore evolutionary mechanisms and the interdependence
entirely of hybrid duplexes; at any point on the chromo- of pathways of macromolecular biosynthesis.
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some, one strand should contain uracil and the sister strand
should contain thymine. From the extent of the DNA ACKNOWLEDGMENTS
synthesis in the deoxyuridine medium, we may conclude
that new rounds of DNA initiation must have occurred We gratefully acknowledge the capable technical assistance of
despite the unavailability of dTMP. If reinitiation had not Laura Bliss and Fred Kung.
occurred, then we should have seen only a 39% increase in This work was supported by research grants MV-205R and
NP770-S from the American Cancer Society.
DNA content (24), representing the average amount of DNA
that would be synthesized between the replication forks and REFERENCES
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clusion is confirmed by the results shown in Table 4. The cations in phage and bacteria. Annu. Rev. Microbiol. 31:473-
new DNA hybridized with plasmid pIT15, which bears a 505.
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