From the Division of Biology, Beckman Research Institute, City of Hope, Duarte, California 91010
Received for publication, December 12, 2002, and in revised form, January 10, 2003
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ABSTRACT |
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UVB mutagenesis is characterized by an abundance
of C The UV component of sunlight is responsible for the induction of
skin tumors, most notably basal cell and squamous cell carcinomas and
most likely also melanomas (1, 2). The most frequent UV-induced
mutations are C The two most abundant UV-induced DNA photoproducts are the
cis-syn cyclobutane pyrimidine dimers
(CPDs)1 and the pyrimidine
(6-4) pyrimidone photoproducts ((6-4)-photoproducts). Most of the
mutagenic and carcinogenic action of sunlight has been attributed to
the UVB portion of the solar spectrum (6), with a possible, but
controversial, role for UVA (320-400 nm) in the induction of melanoma
(7). The CPD is considered the most important UV-induced lesion based
on its relatively high abundance, slow repair, and known mutagenicity
(8-11). In an attempt to dissect the individual contributions of CPDs
and (6-4)-photoproducts to UVB mutagenesis, we have previously
introduced foreign photolyase genes into a mouse cell line that carries
two transgenic mutation reporter genes. We studied the mutations
produced after photoproduct-specific photoreactivation and showed that
the CPD is responsible for a great majority of the mutations induced by
UVB irradiation (11).
Earlier, we and others found that sequences that contain
5-methylcytosine within a dipyrimidine are up to 15 times more
susceptible to CPD formation after exposure to natural sunlight (12) or a UVB light source (13) compared with 254-nm UVC irradiation. Methylation of cytosine enhances CPD formation by sunlight by 5-15-fold (12). This difference may be explained by the higher energy
absorption of 5-methylcytosine compared with cytosine in DNA in the UVB
range at wavelengths between 295 and 320 nm (12, 14). In previous work,
we have examined UV-induced mutational events at methylated CpG
sequences using the lacI and cII transgenes as a
mutational target. Dipyrimidines that contain 5-methylcytosine were
preferential targets for sunlight-induced mutation hot spots (15,
16).
Plasmid constructs containing defined UV photoproducts have been used
to study the mutagenic specificities of CPDs and (6-4)-photoproducts. The mutation frequency obtained with site-specific 5'-TT-CPDs is
generally very low (17-19). This is consistent with the infrequent recovery of mutations at 5'-TT sequences in UV-irradiated cells and is
probably due to the action of DNA polymerase Spontaneous deamination of methylated cytosines is commonly proposed as
the mutational mechanism responsible for C to T transition mutations at
CpG sites in mammalian genes (14, 24, 25). Deamination of cytosine or
5-methylcytosine is expected to occur more rapidly within a CPD as
opposed to within normal double-stranded DNA (26, 27). This process may
play an important role in UV mutagenesis.
We have shown previously that the deamination of cytosine within CPDs
does occur in human cells at a significant rate (28). Here we have
studied the mutagenic consequences that are associated with such
deamination events, in particular with respect to the involvement of
5-methylcytosine.
Cell Lines and Plasmids--
SV-40-transformed DNA
repair-deficient human XP-A fibroblasts (XP12BE) were obtained from the
American Type Culture Collection (Manassas, VA). The cells were grown
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum in a 5% CO2 humidified incubator. The pSP189
plasmid (including a randomly generated 8-base pair signature sequence
at the 3'-end of the supF gene) was kindly provided by
Michael Seidman (29). The E. coli tyrosine amber
suppressor transfer RNA gene, supF, enables read-through of
a UAG stop codon in the lacZ gene, resulting in synthesis of functional Methylation and UVB Irradiation--
The pSP189 plasmid was
methylated in vitro using the CpG-specific DNA methylase
SssI (New England Biolabs, Beverly, MA) according to the
manufacturer's instructions. Control DNA was mock-methylated in the
absence of S-adenosylmethionine. Completion of methylation was confirmed by digestion of an aliquot of the reaction mixture with
the methylation-sensitive restriction endonuclease HpaII. Methylated and unmethylated pSP189 were irradiated in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) with a UVB
source (a Philips TL 20W/12RS lamp filtered through cellulose acetate;
peak emission, 312 nm; lower wavelength cut-off, 295-300 nm) at a dose
of 8000 J/m2 (about 10 min). The UV dose was determined
with a UVX radiometer and a UVB sensor (Ultraviolet Products, Upland,
CA). After UVB exposure, DNA was incubated in TE buffer, pH 7.5, at
37 °C for various time intervals to allow deamination to occur.
Mutagenesis Assay--
The pSP189 shuttle vectors were
transfected into cultures of nucleotide excision repair-deficient human
fibroblasts (XP12BE). Briefly, 5 × 105 cells were
plated into 10-cm tissue culture dishes in Dulbecco's modified
Eagle's medium. Following a 16-h incubation of the cells, a mixture of
plasmid and FuGene 6 transfection reagent (Roche Molecular
Biochemicals) was added. After a 72-h incubation of the cells, the
plasmid was rescued from the human cells by alkaline lysis. Cells were
trypsinized, washed, and resuspended in suspension buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 µg/ml
RNase A), mixed with 1 volume of lysis buffer (0.2 M NaOH,
1% (w/v) SDS), and incubated on ice for 3-5 min, followed by the
addition of neutralization buffer (3 M potassium acetate,
pH 5.5). After a 15-min incubation at room temperature, the mixture was
centrifuged for 10 min at 16,000 × g, and the
supernatant was extracted once with phenol/chloroform. Following
ethanol precipitation, the DNA was resuspended in DpnI
reaction buffer, and unreplicated plasmid was removed by digestion with
DpnI, which recognizes the bacterial adenine methylation
pattern. Then the plasmid was electroporated into MBM7070 bacteria,
which carry a lacZ gene with an amber mutation (29, 30). The transformed bacteria were diluted in 1 ml of SOC medium
and plated on agar plates containing 50 µg/ml ampicillin, 1 µM isopropyl-1-thio- Ligation-mediated PCR (LMPCR)-based Deamination Assay--
For
measuring deamination of cytosines or 5-methylcytosines within
cyclobutane pyrimidine dimers, a sample of the treated plasmids was
used to map deamination rates by LMPCR after a 48- and 96-h incubation
period at 37 °C. The CPDs in UVB-irradiated DNA were repaired to
completion using Escherichia coli CPD photolyase (1 µg of
photolyase for 10 µg of UVB-damaged DNA) in a 100-µl reaction
containing 50 mM Tris-HCl, pH 7.4, 50 mM NaCl,
1 mM EDTA, 10 mM dithiothreitol, 50 µg/ml
bovine serum albumin, 5% glycerol. The photolyase reaction conditions
were as described (31). After phenol/chloroform extraction and ethanol
precipitation, DNA was redissolved in water. The DNA was then incubated
with 1 µg of recombinant His-tagged human MBD4 protein at 37 °C
for 1 h in a 100-µl reaction containing 25 mM Hepes,
pH 7.8, 50 mM KCl, 1 mM EDTA, 2 mM
dithiothreitol. MBD4 converts the uracil and thymine residues in the
DNA, present as U/G or T/G mismatches and originating from cytosine and
methlylated cytosine, respectively, into abasic sites (32, 33). An
excess of MBD4 was used so that mismatches at both CpG and non-CpG
sites were efficiently cleaved (33) (data not shown). After
phenol/chloroform extraction and ethanol precipitation, the DNA was
incubated in 1 M piperidine at 37 °C for 15 min to
convert the abasic sites into DNA strand breaks.
LMPCR was performed on the enzyme-treated DNA as described (34).
Oligonucleotide primers for LMPCR were as follows:
5'-CAAAAAAGGGAATAAGG-3' (supF-1),
5'-CTTAGCTTTCGCTAAGG-3' (supF-4), 5'-TAAGGGCGACACGGAAAT-3' (supF-2), 5'-TCGCTAAGGATCCGGGT-3' (supF-5),
5'-GAAATGTTGAATACTCATACTCTTCC-3' (supF-3),
5'-AAGGATCCGGGTACCGAA-3' (supF-6). In brief, oligonucleotide primer supF-1 or supF-4 was annealed to 0.5 µg
of DNA after enzyme treatment, followed by primer extension of this
primer at 48 °C with Sequenase 2.0 (U.S. Biochemical Corp.). The
oligonucleotide linker, consisting of a 25-mer annealed to an 11-mer
oligonucleotide, was then ligated to the blunt-ended, primer-extended
molecules. After ligation and precipitation of the DNA, gene-specific
DNA fragments were amplified with Taq polymerase (Roche
Molecular Biochemicals) by using the 25-mer of the linker and a
gene-specific PCR primer (primer supF-2 or
supF-5) under conditions given previously (34). After 21 cycles of PCR, the samples were phenol/chloroform-extracted and
ethanol-precipitated, and the amplified fragments were separated on 8%
(w/v) polyacrylamide gels containing 7 M urea. The samples were run until the xylene cyanol dye reached the bottom of the sequencing gel, and the bottom 40 cm of the gel was electroblotted onto
nylon membranes by using an electrotransfer device (Owl Scientific; Cambridge, MA). The sequences were visualized by autoradiography after
hybridization with a single-stranded gene-specific PCR probe. The
hybridization probes were made by repeated run-off polymerization using
primer supF-3 or supF-6 and the respective PCR
products as templates (35). Chemical DNA sequencing reactions (36) were
used with all LMPCRs to provide sequence markers. As a control, the
persistence of CPDs was measured along with the deamination experiments. In this assay, T4 endonuclease V- and photolyase-treated DNA was subjected to LMPCR analysis as described in previous studies (31, 34).
To study the mutational spectrum induced by UVB irradiation in a
CpG-methylated gene, we used the shuttle vector pSP189, which contains
the supF gene as a mutational target. The vector was methylated in vitro at all CpG sequences, using the
CpG-specific DNA methyltransferase SssI. In parallel
experiments, pSP189 was mock-methylated in the absence of
S-adenosylmethionine to produce an unmethylated counterpart.
Completion of the methylation reaction was confirmed by digesting an
aliquot of the reaction mixture with the methylation-sensitive
restriction endonuclease HpaII.
These methylated and unmethylated shuttle vectors were irradiated with
UVB at a dose of 8000 J/m2. At this dose, approximately one
CPD is produced every 0.5-0.8 kilobases. Nonirradiated DNA was used as
a control. After irradiation, an aliquot of the methylated and
unmethylated pSP189 vectors was transfected immediately into nucleotide
excision repair deficient XP-A fibroblasts. The other aliquots of the
UVB-irradiated plasmid were incubated at 37 °C in TE buffer to allow
time for deamination of cytosine and 5-methylcytosine to occur within
CPDs. After the different incubation times, these plasmids were
transfected into XP-A fibroblasts. The cells were allowed to grow for
72 h after transfection. Then the plasmids were rescued, and the
DNA was cleaved with DpnI to remove unreplicated plasmids
still containing the bacterial GATC methylation pattern. Using
HpaII and HhaI digestion, we found that the level
of CpG methylation was largely (>80%) maintained in
DpnI-resistant DNA 72 h after transfection, indicating that there is no active removal of methyl groups from the methylated plasmids and that the methylation pattern is at least partially conserved during DNA replication (data not shown). Conversely, the
unmethylated plasmid did not undergo de novo methylation. The rescued plasmids were then electroporated into MBM7070 bacteria, which carry a lacZ gene with an amber mutation. Plasmids
were isolated from white colonies, and the supF gene was
sequenced. Siblings, indicated by repeated appearance of the signature
sequence, were about 2% of all plasmids and were excluded.
The mutant frequencies in unmethylated and methylated UVB-treated
supF vectors, incubated for different amounts of time
before transfection, were determined (Table
I). The mutant frequency in the absence
of UV irradiation was generally below 1 × 10 T and 5-methylcytosine
T transitions at
dipyrimidine sequences. It is not known how these mutations might
arise. One hypothesis is that UV-induced mutations occur only after
deamination of the cytosine or 5-methylcytosine within the pyrimidine
dimer. It is not clear how methylation of cytosines at the 5-position
influences deamination and how this affects mutagenesis. We have now
conducted experiments with a CpG-methylated supF shuttle
vector that was irradiated with UVB and then incubated at 37 °C to
allow time for deamination before passage through a human cell line to
establish mutations. This led to a significantly increased frequency of CC
TT mutations and of transition mutations at 5'-PymCG-3'
sequences. A spectrum of deaminated cis-syn cyclobutane
pyrimidine dimers in the supF gene was determined using the
mismatch glycosylase activities of MBD4 protein in combination with
ligation-mediated PCR. Methylation at the C-5 position promoted
the deamination of cytosines within cis-syn cyclobutane
pyrimidine dimers, and these two events combined led to a significantly
increased frequency of UVB-induced transition mutations at 5'-PymCG-3'
sequences. Under these conditions, the majority of all supF
mutations were transition mutations at 5'-PymCG-3', and they clustered
at several mutational hot spots. Exactly these types of mutations are
frequently observed in the p53 gene of nonmelanoma skin tumors. This
particular mutagenic pathway may become prevalent under conditions of
inefficient DNA repair and slow proliferation of cells in the human epidermis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
T or CC
TT mutations involving pyrimidine
dinucleotide sequences. These mutations are considered as a
characteristic fingerprint that can be ascribed to solar UV irradiation
(3). Such mutations are often found in the p53 gene of
sunlight-associated skin cancers (4, 5).
, which correctly bypasses these lesions (20, 21). DNA polymerase
is encoded by
the RAD30 gene in yeast and by the XPV
gene in humans. The mutagenicity of a site-specific CPD containing the
5'-TC sequence also is very low, with >95% accurate lesion bypass,
but this has been studied only in Escherichia coli (22).
Studies with yeast mutants indicate that RAD30 may correctly bypass
5'-T-C and 5'-C-C dimers (23). If cytosine-containing CPDs are
indeed bypassed mostly error-free, then what is the origin of C
T mutations at such lesions?
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase. The bacterial strain MBM7070 is cultured on agar containing ampicillin and isopropyl
-D-thiogalactoside, an inducer of
-galactosidase, and
X-gal as a color indicator. Colonies resulting from transformation with
a functional supF gene will be blue, whereas those
containing mutant supF genes will be white.
-D-galactopyranoside,
and 100 µg/ml X-Gal. After an overnight incubation at 37 °C,
wild-type (blue) and mutant (white) colonies were counted to determine
the mutant frequency. Colonies containing a plasmid with a mutated
supF gene were identified, and the plasmids were purified
using Wizard plasmid purification kits (Promega, Madison, WI) and
sequenced. It is unlikely that any significant number of the mutations
will arise in E. coli. DpnI treatment removes the
unreplicated and still lesion-containing plasmids. It should be noted
that the replication of the SV40-based plasmid in human cells is of the
runaway type, and there will be an enormous dilution of the adducted
plasmids. Tens of thousands of progeny molecules are recovered from a
single cell. The plasmid replication cycle is about 15-20 min. Even if
there are a few single cycle progeny, they will be diluted by a huge
excess of nonadducted progeny. In addition, no mutations can be
recovered upon direct transformation of MBM7070 with UV-treated
plasmids (30) because of the high repair capacity of this
strain.2
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3, and
it was not different between unmethylated and methylated targets. After
UVB irradiation and immediate transfection, the mutant frequency
increased to 8-12 × 10
3 (a more than 13-18-fold
increase). However, the mutant frequencies became even higher with
increasing incubation times at 37 °C before transfection (Table I).
At the longest incubation times, the mutant frequencies were almost 30 times higher than in the nonirradiated controls and were over 2 times
higher than in DNA that was transfected immediately after irradiation.
There was no major difference in the mutant frequency trends between
unmethylated and methylated DNA. We confirmed by agarose gel
electrophoresis that no significant DNA degradation had occurred after
a 96-h incubation at 37 °C.
SupF mutant frequencies
Given the increases in mutant frequencies after the pretransfection
incubation, we proceeded to determine the mutational spectra obtained
under various conditions. Although there were few colonies obtained in
the absence of UVB treatment, we sequenced the available mutants (Fig.
1). Most of the mutations were deletions
rather than single nucleotide changes. Further, we selected the samples obtained by immediate transfection and those obtained with the 96-h
preincubation period for the sequencing analysis. This was done
for both the unmethylated and the
methylated plasmids (Figs. 2 and 3).
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As expected from the known mutagenic action of UVB irradiation, most of
the mutations were C T transition events (74-91%), and a sizable
fraction (5-15%) were CC
TT tandem mutations (Table II). When the DNA was incubated at
37 °C for 96 h before transfection, the frequency of tandem
mutations, mostly CC
TT, increased. In the unmethylated samples
immediately transfected, the percentage of tandem mutations was 9.3%
(7 of 75). This value increased to 15.1% (13 of 86) in the sample that
was transfected after 96 h. In the methylated samples immediately
transfected, the percentage of tandem mutations was 6.4% (5 of 78).
This value increased to 14.9% (13 of 87) in the sample that was
transfected after 96 h. Although individually these differences
did not reach statistical significance, the difference did become
statistically significant (p < 0.05;
2
test) when the values from methylated and unmethylated plasmids were
combined (26 of 173 versus 12 of 153 mutants were tandem mutations after delayed transfection). Thus, there is a clear trend
toward an increase in tandem mutations, predominantly of the CC
TT
type, after time is allowed for deamination to occur within CPD
lesions.
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We then focused on the role of CpG methylation in UVB mutagenesis. When
comparing the unmethylated and CpG-methylated mutational targets, there
was a noticeable trend for the mutation hot spots to occur at
dipyrimidines involving 5-methylcytosine (i.e. PymCG trinucleotides). For the unmethylated plasmid, 24% of the mutations involved CpG dinucleotides when the DNA was transfected immediately. This value was 21% when the transfection was done after 96 h
(Table II). A higher percentage of the mutations (36%, or 28 of 78)
were present at methylated CpG sites in the CpG-methylated plasmid. This was particularly striking in the CpG-methylated plasmid that was
transfected after 96 h. In this case, 52% (45 of 87 mutations) involved 5-methylcytosine. Thus, when comparing the unmethylated and
methylated plasmids transfected after 96 h, there is a shift toward a mutational spectrum that is dominated by mutations involving 5-methylcytosines at CpG dinucleotides, and this difference is highly
significant (p < 0.001; 2 test). When
we compared the fraction of mutations at methylated CpGs between the
DNA that was immediately transfected and the one that was incubated for
96 h, the difference was 36% (28 of 78) versus 52%
(45 of 87) (p = 0.05;
2 test).
This difference is also evident when the location of mutational hot spots is considered. In the mutational spectrum of the unmethylated plasmid (Fig. 2), which is similar between the 0- and 96-h
transfection, there is only one moderate mutation hot spot at a CpG
sequence. All of the other hot spots (defined as five or more mutations at the same nucleotide position) involve either CpC or TpC sequences in
which the cytosine is converted to thymine. However, in the mutation spectrum of the methylated supF plasmid transfected
after 96 h (Fig. 3), four of the six mutation hot spots are at CpG sequences.
There may be two independent pathways by which methylation of cytosine
can promote mutagenesis after UVB irradiation. The first one is the
documented enhancement of CPD formation at methylated cytosines in the
UVB range (12, 13). The second pathway may be that 5-methylcytosines
efficiently undergo deamination within CPDs to form dimers containing
thymine. Upon bypass, such lesions would result in C T transitions.
In order to test these possibilities, we have devised an assay that can
measure the deamination of cytosines and 5-methylcytosines within CPDs.
This assay is based on enzymatic photoreversal of CPDs using CPD
photolyase followed by cleavage of the resulting DNA with the
mismatch-specific DNA glycosylase MBD4 (Fig.
4A). MBD4 recognizes T/G as
well as U/G mismatches in DNA (32, 33) and will thus detect the
appearance of deaminated CPDs containing either 5-methylcytosine or
cytosine. An excess of MBD4 was used so that mismatches at both CpG and
non-CpG sites were efficiently cleaved (33) (and data not shown). After
MBD4 cleavage, the DNA was then treated with alkali to produce strand
breaks at the resulting abasic sites. Ligation-mediated PCR was used to
detect and analyze supF sequences (Fig. 4, B and
C). In control lanes, we established that the pattern of T4
endonuclease V-cleavable sites was largely unchanged between the 0-h
samples and the samples incubated for 96 h. Using the photolyase/MBD4 assay, we observed that there was a clear
time-dependent increase in signals with incubation times
proceeding up to 96 h. As expected from the scheme outlined in
Fig. 4A, signals were observed only at cytosines and
5-methylcytosines that are part of a dipyrimidine sequence. Many of the
MBD4-induced signals coincided with mutational hot spots, such as the
ones at nucleotide positions 124, 129, 150, 155, 156, and 163. No
mutations were observed at position 149. The mutational hot spots at
positions 168 and 172 on the upper strand could not be measured by
LMPCR due to the presence of the random signature sequence 3' to the
tRNA gene. However, not all of the strong signals obtained with the
deamination assay, such as the one at position 164, correlated with
mutational hot spots. This is not surprising, since it has often been
difficult to correlate UV damage hot spots in the supF gene
with mutation hot spots (29). Sequence context, perhaps as a
consequence of the abundance of hairpin structures in this tRNA gene,
appears to play an important role in determining which sequence appears as a mutational hot spot. However, it is apparent that in the MBD4
deamination assay, the strongest signals were observed at methylated
CpGs in the UVB-irradiated plasmids incubated for 96 h. Even when
taking into account that a somewhat higher frequency of CPDs was
produced at methylated PymCG sequences in the methylated plasmid, the
extent of deamination seemed to be much higher compared with the
unmethylated plasmid, in particular at sequence positions 156, 159, 163, 155, and 149 (Fig. 4, B and C). The stronger
signals obtained at deaminated 5-methylcytosine-containing dimers are not a consequence of preferential cleavage by MBD4, since this enzyme
equally cleaves or even prefers U/G over T/G mismatches (32,
33).3 At positions 155, 156, and 163, the increased deamination observed with the methylated DNA was
accompanied by an increased C T mutation frequency at the same
nucleotide position (Figs. 3 and 4).
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These data show that 5-methylcytosines efficiently undergo deamination
at 37 °C when they are part of a cyclobutane pyrimidine dimer. The
majority of the deaminated sites correspond to UVB-induced mutational
hot spots.
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DISCUSSION |
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CPDs and (6-4)-photoproducts are the most abundant DNA
photoproducts produced by UVC and UVB irradiation (8, 9). Previous results using in vivo photoenzymatic removal of specific
photoproducts provided evidence that CPDs are responsible for the
majority of UVB-induced mutations in mammalian cells (11). The data
were consistent with a higher level of formation, slower repair, and increased mutagenicity of CPDs compared with (6-4)-photoproducts (8-11). The low mutagenicity of (6-4)-photoproducts in mammalian cells may be a consequence of their low level of induction, efficient repair (although in this study we used an XP-A cell line), and possibly
also a bypass tolerance by DNA polymerase for the most abundant
(6-4)-photoproduct, the one that forms at 5'-TC sequences. DNA
polymerase
preferentially incorporates a guanine opposite the 3'
base of 5'-TT (6-4)-photoproducts, although it is unable to extend
from the inserted nucleotide (37). If, due to structural features of
the lesion or due to deamination of the 3' base, this polymerase would
have the same incorporation specificity opposite the 3'-C of a 5'-TC
(6-4)-photoproduct, then no mutation would be produced.
The mutagenic mechanism involving CPDs is still unclear. T-T dimers,
although induced at high levels, are not very mutagenic. This is
probably a consequence of their correct replication bypass by the
lesion-tolerant enzyme DNA polymerase (20, 21). CPDs containing cytosines, and most notably 5-methylcytosines, are also
abundantly produced in the UVB range (12, 13, 38). Dipyrimidines
containing cytosines, particularly 5'-TC, 5'-TmC, 5'-CC, and 5'-CmC,
are the preferential targets of UV-induced transition mutations in
mammalian cells (8, 9, 39). There are at least two possible pathways
through which CPDs containing cytosines or 5-methylcytosines can cause
mutations (Fig. 5). One pathway involves
direct lesion bypass by a DNA polymerase that incorporates adenines
opposite the C or mC within the dimer. The nature of this polymerase is
unknown. The second pathway includes a model in which the C or mC first
deaminates within the CPD lesion, a reaction that is then followed by
correct bypass during DNA replication. The mutagenicity of a
site-specific CPD containing the 5'-TC sequence in a plasmid construct
is very low, with >95% accurate lesion bypass when studied in
E. coli (22). If cytosine-containing CPDs are bypassed
mostly error-free, then the origin of C
T mutations at such lesions
could be the deamination pathway.
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We and others have proposed that most UV-induced transition mutations
at dipyrimidines containing cytosine may result from correct DNA
polymerase bypass of CPDs containing deaminated cytosine or
5-methylcytosine (16, 28, 40-44). Deamination of cytosine or
5-methylcytosine occurs more rapidly within a CPD as opposed to within
normal double-stranded DNA (26, 27). Deamination of C in T-C or C-C
dimers leads to formation of T-U or U-U dimers, respectively. Adenines
are incorporated with high specificity during bypass of site-specific
T-T, T-U, or U-U dimers in vivo (41, 45). After deamination
of cytosine or 5-methylcytosine within CPDs, DNA polymerase is
probably bypassing these CPDs in an error-free manner (20, 21, 46). The
final outcome would be a C
T or mC
T transition mutation, the
most common type of mutation seen after UV irradiation.
It is not known how DNA damage-tolerant DNA polymerases bypass CPDs
containing nondeaminated cytosines or 5-methylcytosines. One
possibility is that they incorporate adenines (47). However, our
expectation is that they incorporate guanines in a mostly error-free
pathway and that a mutation occurs only after deamination. There is, in
fact, genetic evidence from studies with the yeast homologue showing
that DNA polymerase may bypass CPDs containing cytosine correctly
(23).
We have shown previously that deamination of cytosine in CPDs does occur at significant rates in vivo (28). Here, we have studied the mutagenic consequences of these deamination reactions using a mammalian shuttle vector system. The shuttle vector was either transfected immediately or was incubated for 96 h prior to transfection. With the longer incubation times, the mutant frequencies increased by 2-2.5-fold. The increase was not linear but rather a first order increase, a particularly strong early increase followed by a tendency toward leveling. The reason for this nonlinear increase is not clear, but it could indicate that some of the mutations may have been produced by (6-4)-photoproducts. However, it is likely that deamination reactions would also occur in cells that were transfected immediately and that some of this deamination could occur prior to DNA replication. Thus, with the plasmid transfected immediately after irradiation, we cannot clearly distinguish if the mutations were produced by the deamination pathway or by the pathway of directly replicating cytosine-containing CPDs (Fig. 5). The 2-fold and nonlinear increase in mutant frequency after delayed transfection is consistent with the proposal that most mutations after immediate transfection may also have arisen through the deamination pathway.
We find that incubating the shuttle vector plasmids for an extended
period of time prior to transfection produces two clear effects. The
first is an increase of tandem, mostly CC TT mutations, and the
second is an increased fraction of mutations occurring at
5-methylcytosine bases. How can these two observations be explained? Since both phenomena are dependent on incubating the UVB-irradiated DNA
in vitro at 37 °C, the likely explanation is that
deamination of cytosines within CPDs is causing these effects. We have
confirmed that the deamination reactions do indeed occur, both at
cytosines and at 5-methylcytosines (Fig. 4). The data show that
5-methylcytosines efficiently undergo deamination at 37 °C when part
of a cyclobutane pyrimidine dimer. This is most likely the mechanism
through which UVB irradiation produces mutational hot spots at PymCG
sequences. Several mutational hot spots are seen in the p53 gene in
skin tumors (5, 12). Six of the eight most commonly mutated sites (codons 152, 196, 213, 245, 248, and 282) contain the mutated dipyrimidine in the sequence context 5'-CCG or 5'-TCG. All of these CpG
sequences are methylated to contain 5-methylcytosines in normal human
keratinocytes (48).
In order to explain the increased frequency of CC TT mutations
after in vitro incubation of the plasmid, one can invoke double deamination events in which both cytosines are converted to
uracils (42, 44). The LMPCR assay does not allow the detection of the
two uracils simultaneously, but such events have been detected using a
sensitive PCR assay (28). Since no other explanation seems likely, the
observation of an increased frequency of CC
TT mutations after
in vitro incubation of the plasmid also supports the UV
mutagenesis model involving deamination (Fig. 5). One remarkable finding is the high proportion of CC
TT, and in particular CmC
TT mutations present at CpG sites, in the p53 gene of skin cancers from
xeroderma pigmentosum patients (49, 50). We have not observed a large
number of such mutations in the supF assay. This could be
due to the fact that the major UVB hot spots in the methylated supF gene were at the sequence TCGA, which does not allow
for the formation of such mutations. The high frequency of tandem mutations in UV-exposed skin of xeroderma pigmentosum patients could be
a consequence of complete or almost complete lack of nucleotide
excision repair and slow cell division rates providing enough time for
double deamination events to occur within CPDs. The deamination pathway
of UVB mutagenesis is probably of importance in sun-exposed cells in
the human epidermis.
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ACKNOWLEDGEMENTS |
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We thank Aziz Sancar (University of North Carolina, Chapel Hill, NC) for kindly providing E. coli photolyase, Stephen Lloyd (University of Texas Medical Branch, Galveston, TX) for T4 endonuclease V, Timothy O'Connor (Beckman Research Institute) for recombinant MBD4 protein, and Adrian Bird (University of Edinburgh) for the construct to overexpress human MBD4. Steven Bates is acknowledged for assistance with cell culture work.
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FOOTNOTES |
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* This work was supported by NIEHS, National Institutes of Health, Grant ES06070 (to G. P. P.).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: Division of Biology,
Beckman Research Institute, City of Hope, Duarte, CA 91010. Tel.:
626-301-8853; Fax: 626-930-5366; E-mail: gpfeifer@coh.org.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212696200
2 M. Seidman, personal communication.
3 D.-H. Lee and G. P. Pfeifer, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
CPD, cyclobutane
pyrimidine dimer;
(6-4)-photoproduct, pyrimidine-(6-4) pyrimidone
photoproduct;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
LMPCR, ligation-mediated PCR.
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