(Received for publication, March 16, 1995; and in revised form, July 19, 1995)
From the
The realization that cytosine in cyclobutyl pyrimidine dimers
rapidly deaminates to uracil raised the possibility that this chemical
transformation, rather than an enzymatic polymerase error, is the major
mutagenic step in UV mutagenesis. We have established a sensitive
bioassay system that enabled us to determine the rate of deamination of
cytosine in cyclobutyl pyrimidine dimers in plasmid DNA. This was done
by in vitro UV irradiation and deamination of a plasmid
carrying the cro gene, followed by photoreactivation, and
assaying uracils in DNA by their ability to cause Cro mutations in an indicator strain that was deficient in uracil DNA N-glycosylase. DNA sequence analysis revealed that 27 out of
29 Cro
mutants carried GC
AT transitions, as
expected from deamination of cytosine. Deamination of cytosines in the cro gene in UV-irradiated plasmid pOC2 proceeded at 37 °C
with first-order kinetics, at a rate of (3.9 ± 0.6)
10
s
, corresponding to a
half-life of 5 h. Physiological salt conditions increased the half-life
to 12 h, whereas decreasing the pH increased deamination. The
temperature dependence of the rate constant yielded an activation
energy of 13.6 ± 3.3 kcal/mol. These kinetics data suggest that
deamination of cytosine-containing dimers is too slow to play an
important role in UV mutagenesis in Escherichia coli. However,
it is likely to play an important role in mammalian cells, where the
mutagenic process is slower.
UV radiation produces in DNA a multiplicity of photoproducts, of
which two are believed to be responsible for most of the killing and
mutagenic effects of UV radiation(1) . These are the cyclobutyl
pyrimidine-pyrimidine dimers, and the pyrimidine-pyrimidone 6-4 adducts (2, 3) . Since GC AT transitions constitute a
major class of UV mutations both in prokaryotes and
eukaryotes(4, 5, 6) , cytosine-containing
dimers are likely to be a major pre-mutagenic class of UV lesions.
It is believed that the key step in UV mutagenesis involves a bypass
synthesis reaction, whereby a DNA polymerase replicates through a UV
lesion in DNA (reviewed in (6) and (7) ). According to
this model, the polymerase incorporates with high frequency an
incorrect nucleotide opposite the lesion, a potentially mutagenic
event. The chemical nature of the DNA lesion is a major parameter in
determining the efficiency and specificity of misincorporation opposite
the lesion(1) . Thus, if a UV lesion undergoes a secondary
chemical transformation to yield a different product, this may have a
significant effect on its mutagenicity. This is the case with
cytosine-containing photodimers which undergo spontaneous deamination
to yield uracil-containing photodimers(8) . The special
interest in this reaction stems from the fact that a C U change
is potentially mutagenic, since uracil has the coding properties of
thymine(9) . Thus, if the spontaneous deamination proceeds fast
enough, it adds a component of a non-enzymatic mutagenic reaction to
the process of UV mutagenesis(10) . Clearly, the rate of
deamination is a major factor needed in order to evaluate its
biological importance.
The deamination of normal cytosines in DNA
into uracils is a slow process under physiological conditions. At 37
°C and pH 7.4 it proceeds with a half-life of approximately 200 and
30,000 years in single-stranded and double-stranded DNA,
respectively(11) . This means a deamination of approximately
100 cytosines/human genome/day. However, saturation of the 5,6-bond of
cytosine greatly facilitates the rate of deamination. For example,
5,6-dihydrocytosine deaminates at pH 7.0 and 37 °C with a half-life
of only about 2 h(12) . Since in both types of photodimers the
5,6-double bond is saturated, the rate of deamination of
cytosine-containing pyrimidine dimers is likely to be much higher than
that of cytosine. Previous attempts to determine the rate of
deamination of cytosine-containing cyclobutyl pyrimidine dimers (CPDs) ()led to conflicting results. One set of results suggested
that CPDs deaminate in Escherichia coli with a half-life of
5-6 h(13, 14) , whereas a second set of results
suggested that deamination of CPDs in DNA proceeds with a sharp step
kinetics, and is completed within 55 min in phage
(10) or
in purified phage S13 dsDNA(15) , and within 30 min in ssDNA
purified from phage S13, or in the virion
state(10, 15) . This time difference is critical for
evaluating the significance of deamination, since in E. coli,
for example, mutations are believed to be formed within 30 min after UV
irradiation(6, 16, 17, 18) .
Figure 1: Outline of the experimental scheme for measuring deamination of cyclobutyl dimers in UV-irradiated plasmid DNA.
To assay deamination-induced mutations we used the cro mutagenesis system developed in our laboratory(24) . It is
based on the repressor properties of the cro gene product of
bacteriophage (33) , carried on plasmid pOC2. The tester
strains have a LacZ
LacY
genetic
background and contain a
200ind prophage, in which lacZ is fused to the O
P
operator-promoter of phage
(
200 itself has the
immunity region of phage 21). Upon transformation, the plasmid which
overproduces the Cro repressor is introduced into the tester strain
cells. Under normal conditions, the overproduced repressor binds to the
O
operator and represses expression of lacZ, thus
blocking the production of
-galactosidase. In this case, cells are
unable to ferment lactose and thus give rise to white colonies on
lactose-EMB indicator plates. A mutation that sufficiently reduces the
binding of the repressor to the O
region will cause full or
partial derepression of lacZ, enabling lactose fermentation.
This will lead to a local reduction of pH on the indicator plates and
production of dark red colonies. To avoid loss of the plasmid from the
cells during growth, kanamycin is included in the
plates(19, 24, 30) .
The indicator strains
used were an isogenic pair of Ung and Ung
strains. In order to eliminate any contribution from the SOS
system, we engineered the strain to carry
recA and
umuDC mutations. This inactivates the SOS response in
general(34) , and the SOS-mutagenic pathway in
particular(35, 36) , thus not allowing the processing
of UV lesions into mutations. The conversion of unrepaired GU mispairs
to mutations is unaffected by the lack of the UmuDC and RecA proteins.
As can be seen in Table 2, when an indicator Ung strain was transformed with unirradiated plasmid pOC2, a
background mutation frequency of (0.2 ± 0.1)
10
was observed. A plasmid irradiated with 400
Jm
gave only a marginal increase in mutation
frequency (0.6 ± 0.3)
10
, as expected
from the non-mutability phenotype of the
recA
umuDC indicator strain. When the UV-irradiated plasmid was heated at 37
°C for 5 h, then photoreactivated, and assayed in an
Ung
strain, a dramatic increase of up to 200-fold was
observed (mutation frequency (40.3 ± 8.4)
10
). These mutations were almost entirely dependent
on the combined treatments of UV irradiation, deamination (heating),
and photoreactivation of the plasmid, and on the use of an
Ung
strain.
Mutations arising from deamination of
cytosines should give rise to GC AT transitions. Indeed, DNA
sequence analysis of Cro
mutants revealed that 27 out
of 29 mutants (93%) contained GC
AT transitions, as expected.
The remaining two mutations were GC
CG transversions. We
analyzed also 12 mutants obtained from unirradiated plasmid, and found
8 GC
AT transitions, 2 GC
CG transversions, one deletion,
and one insertion. All GC
AT mutations (in both irradiated and
unirradiated DNA) occurred at cytosines that have one or more adjacent
pyrimidines. For UV-irradiated DNA this is expected for mutations
initiated by a cyclobutyl photodimer. Taken together these results
strongly suggest that our system assays the deamination of
cytosine-containing cyclobutyl pyrimidine dimers, and that this
reaction occurs at 37 °C on a time scale of hours.
Figure 2:
UV dose
dependence of deamination-induced Cro mutations.
Plasmid pOC2 was UV irradiated at the indicated UV doses, after which
it was subjected to deamination by incubation in 10 mM Tris
HCl, 1 mM EDTA (pH 7.5) at 37 °C for 2 h.
The plasmids were then photoreactivated, and assayed for
Cro
mutations in E. coli WBY13TUng
as described under ``Experimental
Procedures.'' Each irradiated plasmid was assayed twice, each time
in four independent transformation tubes. The total colony counts for
the transformations with plasmids irradiated at 0, 50, 100, 150, 200,
and 400 Jm
were 4
10
, 4.5
10
, 5.5
10
, 4.5
10
, 3.9
10
, and 3.8
10
, respectively.
Figure 3:
Kinetics of deamination of cyclobutyl
dimers assayed by the production of Cro mutations.
UV-irradiated plasmid pOC2 (400 Jm
) was subjected to
deamination by incubation in 10 mM Tris
HCl, 1 mM EDTA (pH 7.5) at 37 °C for the indicated periods of time. The
plasmids were then photoreactivated, and assayed for Cro
mutations in parallel in E. coli WBY13TUng
(closed circles) and in E. coli WBY11TUng
(open circles) as described
under ``Experimental Procedures.'' A, kinetics of
mutation formation. B, first-order reaction plot for the
deamination of dimers, based on the data presented in A.
MF
is the mutation frequency at time t,
and MF
is the maximal mutation frequency observed
in our system. See text for details. The 5- and 6-h data points were
not included since they deviated from the earlier points. This is most
likely due to the very high extents of reaction under these conditions
(60% and more of MF
). The resultant rate constant
is (3.9 ± 0.6)
10
s
.
The
deamination reaction is expected to follow first-order kinetics,
described by the rate equation (C*) = (C*)
e
, where (C*)
and (C*)
are the
concentrations of cytosines in pyrimidine dimers before incubation
(time = 0) and after t hours of incubation.
(U*)
, the concentration of uracils arising from the
deamination of the cytosines is (C*)
- (C*)
. After rearrangement the equation becomes,
ln(1-(U*)
/(C*)
) = -kt.
How can U* be calculated from the observed mutation frequencies? Since
the assay detects essentially only mutations at CPD sites, (U*)
is proportional to the mutation frequency at time t (MF
), and the initial concentration of CPDs, (C*)
, is proportional to the maximal mutation
frequency (MF
) observed in our system. Thus, one can assume
that (U*)
/(C*)
=
MF
/MF
. In order to determine MF
we
have conducted several experiments for prolonged incubation times at 42
°C or at pH 6.0, conditions that enhance deamination (see below).
From these experiments, for a plasmid irradiated at 400
Jm
, MF
is (70 ± 14)
10
(see ``Experimental Procedures'').
Thus, the first-order rate constant, k, can be obtained by
plotting ln(1 - MF
/MF
) as a function of
time. Such a plot is shown in Fig. 3B. The rate
constant is (3.9 ± 0.6)
10
s
, implying a half-life of 5 h for the
deamination of CPD in the cro gene.
Figure 4:
Temperature dependence of
deamination-induced Cro mutations. UV-irradiated
plasmid pOC2 (400 Jm
) was subjected to deamination
by incubation in 10 mM Tris
HCl, 1 mM EDTA (pH
7.5) for 1 h at the indicated temperatures. The plasmids were then
photoreactivated, and assayed for Cro
mutations in
parallel in E. coli WBY13TUng
(closedcircles) and in E. coli WBY11TUng
(opencircles) as described under
``Experimental Procedures.'' A, temperature
dependence of mutation formation. B, Arrhenius plot for
determination of the activation energy for deamination of dimers, based
on the data presented in A and in Table 3. The
activation energy calculated from the slope is 13.6 ± 3.3
kcal/mol.
Figure 5:
Kinetics of deamination of cyclobutyl
dimers in the presence of salt assayed by the production of
Cro mutations. UV-irradiated plasmid pOC2 (400
Jm
) was subjected to deamination by incubation in 10
mM Tris
HCl, 1 mM EDTA (pH 7.5), and 0.15 M KCl at 37 °C for the indicated periods of time. The plasmids
were then photoreactivated, and assayed for Cro
mutations in E. coli WBY13TUng
as
described under ``Experimental Procedures.'' A,
kinetics of mutation formation. B, first-order reaction plot
for the deamination of dimers, based on the data presented in A. It yields a rate constant of (1.6 ± 0.2)
10
s
.
The effect of pH on the deamination of CPDs in UV-irradiated plasmid pOC2 was determined at 37 °C. As can be seen (Fig. 6) deamination increased as the pH decreased, with the first-order rate constants varying nearly 50-fold in the tested pH range of 5-9. This implies half-lives of deamination varying from 0.7 h at pH 5 to 34.4 h at pH 9.
Figure 6:
pH dependence of deamination of
cyclobutyl dimers. UV-irradiated plasmid pOC2 (400
Jm) was subjected to deamination by incubation at 37
°C for various time periods in Teorell and Stenhagen's
citrate-phosphate-borate buffer adjusted to the indicated pH values.
The plasmids were then photoreactivated, and assayed for
Cro
mutations in E. coli WBY13TUng
as described under ``Experimental
Procedures.'' The first-order rate constant, k, was
calculated from the mutation frequency data and plotted as a function
of pH.
The degree of fidelity by which DNA polymerases copy damaged nucleotides is critical to the mutagenic effect of DNA lesions. Thus, any secondary chemical reaction that affects the primary DNA lesion might be a major factor in its final mutagenic outcome. This was suggested to be the case for UV light that produces two major types of DNA lesions: the cyclobutyl pyrimidine dimers, and the 6-4 pyrimidine-pyrimidone adducts. The TT cyclobutyl dimers are stable in DNA, and are not known to undergo any further chemical transformation. However, it was noticed in the early days of nucleic acid photochemistry that cytosines in cyclobutyl dimers were rapidly deaminated to uracils(8) . This occurs generally in cytosine derivatives in which the 5`-6` bond is saturated. Since uracil has the coding properties of thymine, this secondary chemical reaction is potentially mutagenic.
Tessman and co-workers (10, 15) have proposed that the major mutagenic step in UV mutagenesis is not due to a polymerase error, but rather due to deamination of CPDs, followed by accurate replication of the uracil-containing dimers. This suggestion is consistent with the observation that site-specific TT and UT cyclobutyl photodimers in M13 ssDNA were copied with relatively high fidelity in E. coli in vivo(39, 40) , however, it is not clear whether it provides a general explanation for chromosomal UV mutagenesis in E. coli(41) . Clearly, a critical parameter in assessing the importance of deamination in UV mutagenesis is the rate of deamination of pyrimidine dimers in DNA.
The assay system that we
have developed measures in vitro deamination of CPDs in
UV-irradiated plasmid DNA utilizing a mutagenesis bioassay. The
presence of uracils produced by the deamination reaction was assayed by
their ability to cause GC AT transitions in the cro reporter gene. This is a sensitive bioassay that enabled us to
determine rates of deamination in a biologically active DNA molecule,
under physiological temperature and salt conditions.
In our assay
system deamination of CPDs follows first-order kinetics, as expected,
with a rate constant of (3.9 ± 0.6) 10
s
at 37 °C in 10 mM Tris
HCl, 1 mM EDTA (pH 7.5), corresponding to a
half-life of 5 h. This result is in agreement with the deamination rate
in E. coli in vivo, evaluated to be 2.2
10
min
(half-life 5.3
h(14) ). Our results differ from the results of Tessman and
co-workers, who reported a sharp step kinetics for the deamination of
both ssDNA and dsDNA, with deamination completed within 55 min in
dsDNAs from phages
(10) or S13(15) , and within
29 min in ssDNA from phage S13(10, 15) . We do not
know the reason for this difference, especially since some of the
experiments were performed under similar buffer and temperature
conditions.
Extensive chemical studies were performed on the
deamination of defined synthetic dinucleotide cyclobutyl dimers. As can
be seen in Table 4, in all cases studied, a first-order
deamination kinetics was observed, and the rate constants were close to
the one calculated based on our bioassay. For example, the half-life of
the cis-syn cyclobutyl dimer of pdCpdT in 10 mM phosphate buffer at pH 7.0 was 6.8 h at room temperature (42) (Table 4). Since the reaction conditions were not
identical in the different studies, the results are not fully
comparable. However, the emerging consensus from the data presented in Table 4is that deamination of CPDs in DNA proceeds by first-order
kinetics, and with a half-life of several hours, depending on
conditions. It is noteworthy that one set of conditions (10 mM TrisHCl, 1 mM EDTA, pH 7.5, 37 °C) gave a
half-life of 5 h, similar to that evaluated for E. coli in
vivo(14) . The activation energy for the deamination of
CPDs in DNA calculated based on the bioassay gave a value of 13.6
± 3.3 kcal/mol, remarkably similar to the activation energy of
the cis-syn isomer of the cyclobutyl dimer of the dinucleotide
dpTdpC, which was determined to be 13.7 kcal/mol(43) . These
two values are within the error margin of the activation energy of 17
± 3 kcal/mol estimated based on analysis of thermal dependence
of UV mutations in glutamine tRNA in E. coli in
vivo(44) .
What is the significance of our data to in vivo UV mutagenesis? The rates of deamination are affected primarily by salt conditions and pH. Under physiological salt conditions (0.15 M KCl or 0.15 M potassium glutamate), deamination was slowed down to a half-life of 12 h. We have not determined the rate of deamination in ssDNA. However, if the dinucleotides are taken as a model system for ssDNA (Table 4), one can anticipate that deamination rates of dimers in ssDNA will not be much different from dsDNA. Another factor that may influence deamination rate is the pH (Fig. 6). In the cell this may be caused by local interactions with proteins or other molecules, and thus alter locally deamination rates.
The 6-4 adducts could not be
assayed in our system since they are not photoreactivated by DNA
photolyase. However, they do not interfere with our assay since they
are most likely repaired by the UvrABC nucleotide excision repair in
the indicator strain. The concomitant removal of uracils originating
from CPD's by the repair of 6-4 adducts is not likely to be
significant since the average interlesion distance in the UV-irradiated
plasmid is longer than 500 nucleotides (20 or less lesions in a
5-kilobase pair plasmid), whereas the repair patches are of the order
of 10-30 nucleotides(1) . Any remaining 6-4 adducts will
not give rise to mutations due to the recA
umuDC genetic background of the indicator strain. Studies on the
deamination of the 6-4 adduct in the dinucleotide dCpT have shown that
it deaminates with a half-life of 152-413 h (depending on
conditions), nearly 2 orders of magnitude slower than
CPDs(42) . Extrapolating to DNA, this suggests that the
deamination of the 6-4 adducts is unlikely to be a significant process
under physiological conditions.
In E. coli UV mutations are believed to be fixed within 30 min(16, 17, 18) . Thus, our results lead us to conclude that deamination of pyrimidine dimers does not play a significant role in UV mutagenesis in E. coli. On the other hand, it is likely to be a significant factor in mammalian cells, where mutagenesis, repair, and replication are much slower(1) .