(Received for publication, August 22, 1995; and in revised form, February 10, 1996)
From the
To investigate the effect of the major UV-induced lesions on
SV40 origin-dependent DNA replication and mutagenesis in a mammalian
cell extract, double-stranded plasmids containing a single cis,syn-cyclobutane dimer or a pyrimidine-pyrimidone(6-4)
photoproduct at a unique TT sequence have been constructed. These
plasmids have been used as templates in DNA replication-competent
extracts from human HeLa cells. Plasmids containing a single pyrimidine
cyclobutane dimer on the potential lagging strand for DNA replication
are replicated with an efficiency approximately equal to that of an
unmodified plasmid. A small decrease in replication efficiency of
20% was observed when the lesion was located on the potential
leading strand for DNA replication. In both orientations, DpnI-resistant, replicated closed circular plasmid DNA was
sensitive to nicking by the pyrimidine dimer-specific enzyme, T4
endonuclease V, indicating that complete replication of the damaged
plasmid occurs in vitro. In contrast, a(6-4) photoproduct,
within the same site and sequence context on the lagging strand for DNA
synthesis, inhibits replication in vitro by an average of
50%, indicating that the mammalian replication complex responds
differently to the two major UV-induced lesions during DNA replication in vitro. Analysis of the DpnI-resistant, replicated
DNA for mutations targeted to the lesion site indicates that neither of
these lesions resulted in significant mutagenesis. UV-induced lesions
at TT sites may therefore be poorly mutagenic under these conditions
for DNA replication in human cell extracts in vitro.
Exposure to the ultraviolet component of sunlight is a
significant risk factor in the development of skin cancers, especially
squamous cell carcinomas and, to a lesser extent,
melanomas(1) . While the mechanisms underlying the development
of these cancers are complex, it has been observed that skin cancer
cells from both humans (2) and rodents (3) show base
changes in the sequences of critical genes, such as the p53 tumor
suppressor gene, that have the characteristics of UV-induced mutations.
In particular, the mutations in these genes are primarily at
dipyrimidine sequences, potential sites of UV-induced DNA damage. The
occurrence of tandem double-base CC TT transitions is also more
common in mutated p53 genes from skin tumors than in mutated p53 from
tumors of internal organs(4) . These results indicate that skin
carcinogenesis may involve the conversion of UV-induced genomic DNA
damage into base changes during the process of replication of damaged
DNA. However, the molecular mechanisms by which damaged DNA is
replicated and mutations are fixed are not well understood.
The
major types of DNA damage induced by UV irradiation occur at
dipyrimidine sequences; these are cyclobutane pyrimidine dimers and
pyrimidine-pyrimidone(6-4) ()photoproducts. A smaller
proportion of thymine and cytosine hydrates is also formed (5) . Thus, random UV irradiation of plasmid DNA or of cells in
culture leads to the formation of a variety of photoproducts involving
thymines, thymine and cytosine, and cytosines. The relative
contribution of the various lesions to the inhibition of DNA synthesis
and to mutagenesis has therefore been difficult to determine. In
addition, studies using cultured cells may be complicated by the
effects of DNA repair, which can vary in a site- and DNA
strand-specific manner(6, 7) .
UV irradiation of
mammalian cells leads to a transient inhibition of DNA
synthesis(8, 9, 10) , which may allow time
for the repair of potentially mutagenic lesions in the template before
replication proceeds(11) . While it has been proposed that this
inhibition of DNA synthesis is due to the blockage of replication forks
at the sites of DNA damage(12, 13) , there is also
evidence for inducible responses that are involved in the inhibition of
initiation of new origins of replication(14, 15) . In
addition, DNA polymerase activity may also be inhibited by direct means
following irradiation of cells, for example by the induction of the p21
protein, which results in the inhibition of processive DNA synthesis by
DNA polymerase (16) .
To directly determine the effect of a single UV-induced lesion in DNA on the mammalian replication complex, we have constructed plasmid molecules that contain either a single cis,syn-cyclobutane pyrimidine dimer or a(6-4) photoproduct at a unique TT site and used these plasmids as templates for DNA replication in vitro in extracts from human HeLa cells. It has been shown previously that mutagenic replication of randomly UV-damaged DNA occurs in these extracts(17, 18) . By utilizing specifically modified plasmid derivatives, we demonstrate that complete replication past a UV-induced lesion occurs in vitro. Previous studies using site-specifically modified constructs in bacterial (19, 20, 21) and yeast (22, 23) systems have shown that these UV-induced lesions can differ substantially in their mutagenic potential. The cell-free approach described here allows the effect of specific lesions on DNA replication and mutagenesis to be studied under conditions in which the complete replication complex is active rather than individual DNA polymerases and repair of these lesions is essentially absent(17) . Using this approach, we show that the mammalian cell replication complex responds differently to a pyrimidine dimer and a(6-4) photoproduct at the same site and sequence context, with the(6-4) photoproduct being substantially more inhibitory to DNA replication in vitro that the pyrimidine dimer. However, neither lesion results in significant mutagenesis at the TT site following DNA replication in vitro.
Figure 1:
Construction of plasmids
containing a cyclobutane pyrimidine dimer or a
pyrimidine-pyrimidone(6-4) photoproduct at a unique TT site. Following
digestion of the plasmid pZ189 with EcoRI, dephosphorylation
of the EcoRI ends, and digestion with AatII, the
large fragment (5430 base pairs) was isolated by electroelution from an
agarose gel. This fragment had a ligatable AatII site and a
non-ligatable EcoRI site. Unmodified or modified linker (shown
by a line over the TT site) was ligated to this fragment
between the EcoRI and AatII sites in two steps. Ligation 1 was carried out at a total DNA concentration of 20
ng/µl and resulted in ligation of the AatII sites of the
vector and linker. The EcoRI ends of this molecule were
5`-phosphorylated using T4 polynucleotide kinase, and Ligation 2 was carried out at a total DNA concentration of 0.2 ng/µl. The
resulting closed circular molecules were isolated by electroelution
from an agarose gel. To construct a modified plasmid having the lesion
in the opposite orientation for replication, the EcoRI-AatII fragment of plasmid pZ189R2, in which the
orientation of the sequences between the BamHI sites,
containing the bacterial -lactamase, supF, and pBR327
origin, are reversed relative to the SV40 origin of replication, was
used as the vector sequence.
The EcoRI-AatII fragment of pZ189 or pZ189R2 was prepared by digesting the plasmid with EcoRI, followed by dephosphorylation using calf intestinal alkaline phosphatase. The dephosphorylated molecule was then digested with AatII, and the large fragment (5430 base pairs) was purified by electroelution following electrophoresis on a 1% agarose gel. Modified double-stranded linker DNA was prepared as follows. The HPLC-purified 11-mer, either unmodified or containing a single cyclobutane pyrimidine dimer or(6-4) photoproduct at the TT site, was phosphorylated at the 5`-end using T4 polynucleotide kinase. Following removal of the kinase by centrifugation through a Centricon 30 microconcentrator, the 11-mer was recovered in the filtrate and concentrated by centrifugation using a Centricon 3 microconcentrator. The phosphorylated 11-mer was then annealed to the unphosphorylated 19-mer 5`-AATTCTCCAACTTGCACGT-3`. The 19-mer was synthesized on an Applied Biosystems oligonucleotide synthesizer and subsequently purified by electroelution from a 16% polyacrylamide gel using standard techniques(28) . Annealing of the 11-mer to the 19-mer was carried out by heating to 75 °C for 10 min, followed by slow cooling to room temperature.
Ligation of the double-stranded linker to either of the EcoRI-AatII fragments was carried out in two steps at 12 °C (see Fig. 1). The first ligation, carried out using equimolar amounts of vector and linker DNA at a total DNA concentration of 20 ng/µl, resulted in ligation of the linker to the vector fragment (see Fig. 1, Ligation 1). The EcoRI ends were rephosphorylated using T4 polynucleotide kinase, and a second ligation was carried out at a DNA concentration of 0.2 ng/µl (see Fig. 1, Ligation 2). During this reaction, intramolecular ligation resulted in the formation of completely closed circular plasmid molecules. The reaction products were separated by electrophoresis on a 1% agarose gel in the presence of 5 µg/ml ethidium bromide. The position of the completely closed circular DNA was identified by comparison with the mobility of a small aliquot of the ligation mixture run in a separate lane on the same gel and visualized by UV transillumination. The preparative DNA samples were not exposed to UV light. Completely closed circular molecules were isolated from the gel by electroelution and purified by spin dialysis against 10 mM Tris, 1 mM EDTA (pH 7.6), followed by two rounds of extraction with phenol and with chloroform, and ethanol precipitation. Following extraction with water-saturated butanol to remove any residual ethidium bromide, the DNA was recovered by precipitation with ethanol and resuspended in 10 mM Tris (pH 7.6), 1 mM EDTA.
The exact position of the
lesion in the plasmid construct was also mapped by primer extension
analysis. UV-induced lesions in the DNA template block DNA synthesis by Taq DNA polymerase during the polymerase chain
reaction(29) . Construct DNA was incubated with 2.5 units of Taq polymerase in 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl, 0.1% Triton X-100, 0.01%
gelatin, and 200 µM dNTP, with 0.3 pmol of the
5`-
P-labeled primer 5`-TAATGCTTTTACTGG-3`, which anneals
81 bases 3` to the potential lesion site. Linear amplification was
carried out in the polymerase chain reaction for 40 cycles of 30 s each
at 95, 45, and 72 °C. The products were analyzed by electrophoresis
on a 6% denaturing polyacrylamide gel, followed by autoradiography of
the dried gel. A second method was also used to confirm the position of
the pyrimidine cyclobutane dimer in the plasmid construct. Plasmid DNA
was digested with the single-cut enzyme BsaHI, which cuts at a
position 388 base pairs from the lesion site. The linearized DNA was
labeled at the 5`-end with [
-
P]ATP using T4
polynucleotide kinase. Following treatment with T4 endonuclease V,
formation of a 388-base fragment was analyzed by 6% denaturing
polyacrylamide gel electrophoresis and detected by autoradiography of
the dried gel.
To detect the presence of the(6-4) photoproduct in
the plasmid construct, the DNA was first linearized by treatment with BsaHI and then labeled at the 5`-end with
[-
P]ATP. The 5`-labeled DNA was either left
untreated or treated with T4 endonuclease V as described or with 1 M piperidine for 30 min at 90 °C. The DNA was recovered by
ethanol precipitation, and any remaining piperidine was removed by
drying overnight in a Savant SpeedVac vacuum evaporator. DNA fragments
were detected by denaturing polyacrylamide gel electrophoresis,
followed by autoradiography of the dried gel. The presence of a(6-4)
lesion in the DNA resulted in the formation of a
P-labeled
388-base fragment following piperidine treatment.
Figure 2:
Analysis of the pyrimidine
dimer-containing plasmid DNA. A, constructs, prepared as
outlined in Fig. 1and purified by electroelution, were analyzed
by agarose gel electrophoresis in the presence of 5 µg/ml ethidium
bromide. Form IV, completely closed circular plasmid DNA; M, pZ189 marker plasmid; N, plasmid constructed using
an unmodified linker; CPD, plasmid constructed using a TT
pyrimidine dimer-containing linker. B, unmodified and
pyrimidine dimer-containing constructs were treated with T4
endonuclease V (T4 endo V) at 37 °C for 2 h. The DNA was
purified by extraction with phenol, followed by ethanol precipitation.
DNA was analyzed by electrophoresis on a 1% agarose gel in the presence
of 5 µg/ml ethidium bromide. N, unmodified plasmid; CPD, pyrimidine dimer-containing plasmid. C,
unmodified and pyrimidine dimer-containing constructs were analyzed by
polymerase chain reaction using the 5`-P-labeled primer
5`-TAATGCTTTTACTGG-3` and Taq DNA polymerase. The primer
anneals 81 bases 3` to the lesion site. To map the position of the
lesion in the construct sequence, extension products derived from the
unmodified construct (N) and from the pyrimidine
dimer-containing construct (CPD) were analyzed on a 6%
denaturing polyacrylamide gel alongside a sequencing ladder (right
panel; in the order A, T, G, C) generated from the same plasmid
using this primer. Full-length and arrested DNA products (arrows) and the plasmid DNA sequence were detected by
autoradiography of the dried gel. The DNA sequence around the lesion
site is shown on the right; the position of the lesion is
indicated by asterisks.
Figure 3:
Analysis of(6-4) photoproduct-containing
plasmid DNA. A, completely closed circular (Form IV) plasmid
DNA, prepared following the ligation protocol outlined in Fig. 1, was isolated by electroelution from a 1% agarose gel.
The isolated DNA was analyzed by electrophoresis on a 1% gel containing
5 µg/ml ethidium bromide, followed by UV transillumination and
photography. M, pZ189 marker plasmid; N, plasmid
constructed using the unmodified oligonucleotide fraction; (6-4), plasmid constructed using the(6-4)
photoproduct-containing fraction. B, the presence of a(6-4)
photoproduct in the purified DNA was determined. Unmodified and(6-4)
photoproduct-containing construct DNAs were linearized using BsaHI and labeled at the 5`-end with P.
Unmodified (lanes 1, 3, and 5) and(6-4)
lesion-containing (lanes 2, 4, and 6) DNA
samples were then processed in parallel reactions and either left
untreated (lanes 1 and 2) or treated with 1 M piperidine (lanes 3 and 4) or with T4
endonuclease V (T4 endo V; lanes 5 and 6).
As a control for T4 endonuclease V activity, unmodified (lanes 7 and 9) and pyrimidine dimer-containing (lanes 8 and 10) construct DNAs were also treated with enzyme
buffer (lanes 7 and 8) or with T4 endonuclease V (lanes 9 and 10). DNA was then separated by
electrophoresis on a 6% polyacrylamide gel, followed by autoradiography
of the dried gel. M, 5`-
P-labeled 123-base pair
marker. Numbers on the left refer to the sizes of the marker
fragments in bases. The arrow indicates the position of the
388-base fragment generated by cleavage of the DNA at the site of
the(6-4) photoproduct or the cyclobutane pyrimidine
dimer.
In contrast, the construct prepared using the modified linker was sensitive to piperidine treatment as indicated by the presence of a band of 388 bases on the autoradiogram (Fig. 3B, lane 4). While the fact that piperidine treatment resulted in degradation of a large proportion of both the unmodified and(6-4) photoproduct-adducted DNAs (Fig. 3B, lanes 3 and 4) does not allow an estimate of the percent of molecules that contain the(6-4) photoproduct to be made, the data indicate that the lesion that is present in the construct is primarily a(6-4) photoproduct rather than a cyclobutane pyrimidine dimer. Analysis of the(6-4)-containing construct by the linear polymerase chain reaction method also indicated the presence of an adduct at the predicted site (data not shown).
Figure 4:
Kinetics of replication of specifically
modified constructs in vitro. DNA replication reactions were
carried out as described under ``Experimental Procedures''
using unmodified plasmid, pyrimidine dimer-containing plasmid, or(6-4)
photoproduct-containing plasmid as the template in the presence and
absence of SV40 large T antigen. At the times indicated following
incubation at 37 °C, an aliquot was withdrawn from each reaction,
and the extent of incorporation of radioactivity from
[-
P]dCTP into trichloroacetic
acid-precipitable material was determined by liquid scintillation
counting.
and
, unmodified plasmid;
and
,
dimer-containing plasmid;
and
, (6-4)
photoproduct-containing plasmid. Closed symbols indicate
incorporation in the presence of large T antigen; open symbols indicate incorporation in its absence.
In the absence of SV40 large T antigen in the reaction, the incorporation of radioactivity was low, indicating that DNA repair-type synthesis is not occurring to a significant extent, consistent with previous observations on UV-irradiated DNA incubated in this type of cell extract ( (17) and Fig. 4). To examine the replication reaction in more detail, the DNA products were purified and analyzed by agarose gel electrophoresis in the presence of 5 µg/ml ethidium bromide, followed by autoradiography of the dried gel. The results indicate that in the absence of large T antigen, a low background of radioactivity is incorporated into both the unmodified and dimer-containing Form I DNAs (Fig. 5A, first and third lanes). The majority of the incorporation of radioactivity into the plasmid DNA was dependent on the presence of large T antigen in the reaction (Fig. 5A, second and fourth lanes), indicating that this represents DNA replication. To test whether this was the case, the products of DNA replication reactions carried out in the absence or presence of large T antigen were treated with the enzyme DpnI. Unreplicated input plasmid DNA, which carries the bacterial methylation pattern at 5`-GATC-3` sequences, is sensitive to digestion by this enzyme. In contrast, any DNA that becomes completely replicated in vitro is resistant to digestion since the mammalian cell extract lacks the enzymes required for methylation of the newly synthesized strand. While treatment of the replicated DNA with DpnI and the subsequent processing resulted in some conversion of closed circular (Form I) DNA to nicked circular (Form II) DNA (Fig. 5B, second and fourth lanes), the majority of the DNA labeled in the presence of T antigen was resistant to digestion by the enzyme, consistent with the conclusion that this synthesis represents replication rather than repair-type synthesis (Fig. 5B). The low level of labeled DNA observed with both constructs in the absence of T antigen was sensitive to digestion by DpnI, indicating it represents background incorporation and not complete plasmid replication (Fig. 5B, first and third lanes).
Figure 5: Resistance of replicated pyrimidine dimer-containing DNA to DpnI digestion. A, plasmid DNA (pZ189-TT), either unmodified (N) or containing a single pyrimidine dimer (CPD), was incubated under the conditions for DNA replication in vitro for 180 min in the presence or absence of SV40 large T antigen (Tag). The DNA was purified from the reaction, and the products were analyzed by electrophoresis on a 1% agarose gel in the presence of 5 µg/ml ethidium bromide. The labeled DNA was detected by autoradiography of the dried gel. The positions of supercoiled (Form I) and nicked circular (Form II) DNAs following electrophoresis are indicated. B, the products of DNA replication in the presence and absence of large T antigen, shown in A, were incubated with 0.3 units of the enzyme DpnI (+) or with enzyme buffer only (-) at 37 °C for 1 h. The DNA was purified from the reaction by extraction with phenol and with chloroform, followed by ethanol precipitation, and analyzed on an agarose gel as described for A. The positions of labeled Form I and II DNAs are shown. The DNA fragments that migrate faster than Form I DNA result from DpnI digestion of plasmid labeled in a large T antigen-independent manner.
Figure 6: Sensitivity of DpnI-resistant, replicated dimer-containing plasmid DNA to nicking by T4 endonuclease V. A, plasmid DNA (pZ189-TT), either unmodified (N) or containing a single pyrimidine dimer (CPD) on the lagging strand for DNA replication, was incubated for 180 min under the conditions for in vitro DNA replication in the presence of SV40 large T antigen. Following purification of the DNA from the reaction, the products were treated with DpnI at 37 °C for 1 h. Each DpnI-treated DNA was divided into two aliquots and incubated with either T4 endonuclease V (T4 endo V; +) or enzyme buffer(-) for 2 h at 37 °C. The products were purified by phenol extraction and ethanol precipitation and analyzed by electrophoresis on a 1% agarose gel in the presence of 5 µg/ml ethidium bromide. Labeled DNA bands were detected by autoradiography of the dried gel. B, conditions were the same as those described for A, except that the plasmid DNA (pZ189R2-TT) carried the TT dimer on the potential leading strand for DNA replication. Following replication in vitro, the DNA was treated first with DpnI and then with T4 endonuclease V as described for A. N, unmodified plasmid; CPD, dimer-containing plasmid; -, without T4 endonuclease treatment; +, with T4 endonuclease V treatment. C, the DpnI-treated unmodified (N) and dimer-containing (CPD) plasmids were treated with the enzyme BglI, and the products were analyzed by electrophoresis on a 1% agarose gel. Restriction fragments were detected by autoradiography of the dried gel. Numbers refer to the sizes of the fragments in base pairs. Forward, forward orientation, in which the dimer is on the lagging strand for replication (A); Reverse, reverse orientation, in which the dimer is on the leading strand for DNA replication (B).
To examine whether replication in vitro was influenced by
the position of the adduct on the leading or lagging strand for DNA
replication, a construct in which the pyrimidine dimer adduct was
placed in the opposite orientation relative to the SV40 origin of
replication (in pZ189R2-TT), such that it would be on the potential
leading strand for DNA replication, was also used as a template. When
using this template, the extent of replication, as determined from the
level of incorporation of radioactivity from
[-
P]dCTP into trichloroacetic
acid-precipitable material, was
80% of that of the unmodified
construct. While this is a small difference, these results suggest that
a single pyrimidine dimer may have a slightly greater inhibitory effect
on DNA replication when located on the leading strand for DNA
replication compared to when it is located on the lagging strand.
However, a more detailed analysis of the replication of plasmids in
which the dimer is located at different positions relative to the SV40
origin of replication will be required to fully elucidate the effect of
this lesion on leading versus lagging strand DNA replication.
The identity of the DpnI-resistant, replicated DNA was confirmed by digestion of the replicated DNA with the enzyme BglI, which generates fragments of 4396 and 1048 base pairs in the normal orientation (in which the lesion is on the potential lagging strand) (Fig. 6C, first and second lanes), and of 3406 and 2038 base pairs in the reverse orientation (in which the lesion is on the potential leading strand) (Fig. 6C, third and fourth lanes).
To determine if the plasmid containing the pyrimidine dimer on the
potential leading strand also became completely replicated, DpnI-treated DNA was treated with T4 endonuclease V and
analyzed as described above for the lagging strand construct.
Densitometric analysis of the relative amount of labeled Form I DNA
indicated that 59% of the replicated Form I DNA is sensitive to
nicking (Fig. 5B, compare third and fourth
lanes). While this value is somewhat higher than the value
obtained with the lagging strand construct (45%; see above), in both
cases, the value is close to 50%, which would be expected if a single
round of replication occurred on both the undamaged and adducted
strands. However, although small differences between the extent of
replication of the damaged strand in these two constructs may be
revealed by further analysis, the observation that the relative amount
of T4 endonuclease V-sensitive Form I DNA (Fig. 5, A, third and fourth lanes; and B, third and fourth lanes) is close to 50% in both cases suggests
that both the undamaged and dimer-containing strands are replicated in
both orientations. The results obtained using these two constructs,
which contain a single cyclobutane pyrimidine dimer in the same
sequence context, but with a different orientation relative to the
origin of replication, suggest that this lesion does not significantly
block DNA replication when located on the lagging strand under these
conditions in HeLa cell extracts in vitro. When the lesion is
on the putative leading strand, a small reduction in replication
efficiency was observed, which could suggest that this lesion has a
more pronounced inhibitory effect on leading strand replication
compared with replication on the lagging strand.
Figure 7: Replication and DpnI resistance of DNA containing a single(6-4) photoproduct in vitro. A, unmodified (first three lanes) or (6-4)-containing (last three lanes) constructs were incubated in parallel reactions under in vitro DNA replication conditions either in the absence(-) or presence (+) of SV40 large T antigen (Tag). The reactions with T antigen (second and third lanes and fifth and sixth lanes) represent duplicate reactions run at the same time. Product DNA was purified, and equal proportions of each DNA sample were electrophoresed on a 1% agarose gel containing 5 µg/ml ethidium bromide. Labeled supercoiled (Form I) and nicked circular (Form II) DNAs were detected by autoradiography. B, plasmid DNA containing a single(6-4) photoproduct was incubated under DNA replication conditions in the absence or presence of large T antigen. The DNA was purified from the reaction, and labeled Form I and II DNAs were separated by electrophoresis on an agarose gel and detected by autoradiography (left panel). An aliquot of this DNA was treated with DpnI (as described in the legend to Fig. 5), and the products were analyzed by electrophoresis on a second agarose gel (right panel). Labeled DNA was detected by autoradiography of the dried gel.
Figure 8:
Hybridization analysis of construct DNA
replicated in vitro. Following DNA replication in
vitro, plasmid DNA was treated with DpnI to remove
unreplicated DNA and then with dam methylase. E. coli MBM7070 was transformed with the treated products and plated on LB
plates containing 50 µg/ml ampicillin. The resulting transformants
were transferred to a second LB plate using a sterile toothpick. The
colonies were then transferred to a nylon membrane and hybridized to
the 5`-P-labeled wild-type 19-mer sequence
(5`-AATTCTCCAACTTGCACGT-3`). After washing at room temperature and then
at the selective temperature of 56 °C, nonhybridizing colonies were
detected by autoradiography of the membranes. A representative
autoradiogram of a washed membrane is shown. The top row of
colonies represents control plasmids that were used to transform E.
coli MBM7070 directly, without replication in vitro,
having a wild-type TT, CT, or CC sequence at the potential lesion site. Outlined colonies in the top row were transformed by
the CT- and CC-containing plasmids, which failed to hybridize at this
temperature. The order of the plasmids used to transform the colonies
in the top row is as follows: TT, CT, CC, TT, CT, and CC. The outlined colony in the sixth row represents a
nonhybridizing colony obtained following transformation of the tester
strain with DpnI-treated, replicated DNA containing the
cyclobutane pyrimidine dimer.
In the case of the plasmid containing the
pyrimidine cyclobutane dimer on the lagging strand, 3046 transformants
were screened in total from three experiments, and 138 nonhybridizing
colonies (4.5%) were identified. Sequence analysis of plasmid DNA
isolated from 22 of these colonies revealed that 21 of 22 (95%) of
these plasmids were construction-related alterations, which lacked the
19-mer insert, contained more than one 19-mer insert, or had a base
change in the AatII ligation site. The observed failure of
plasmids containing multiple inserts to hybridize may possibly be due
to the formation of a hairpin structure in this region during the
washing process, which prevents the 19-mer from annealing. One plasmid,
which did not belong to any of the above classes, contained an A
T transversion within the insert sequence, but that was not targeted to
the lesion site. These data indicate that the frequency of targeted
mutations is <0.2%, assuming that >95% of the total
nonhybridizing colonies represent construction-related alterations.
This construction-related background frequency is similar to that
reported previously for the construction of single-stranded vectors
using this specifically modified
oligonucleotide(19, 20, 21, 22, 23) .
Plasmid DNA was also prepared from 40 randomly chosen colonies that
gave a positive hybridization signal under these conditions. These
plasmids all carried the wild-type 19-mer sequence (data not shown),
making it unlikely that the screening process failed to detect plasmids
carrying mutations at the lesion site.
In the case of the(6-4)
photoproduct-containing plasmid, a total of 3050 colonies were
screened, resulting in 88 nonhybridizing (2.9%) colonies. Sequence
analysis of 45 colonies revealed that 41 of 45 (91%) belonged to the
background categories listed above. Of the remaining four, one had a
single-base deletion (TT T) targeted to the lesion site. The
other three comprised A
C and A
T transversions at
position 4 in the 11-mer sequence (5`-GCAAGTTGGAG-3`) and a deletion of
G at position 5 in this sequence. Assuming that 91% of the
nonhybridizing colonies were of the background type, the mutation
frequency with this plasmid was <0.3%. These numbers of mutations
are too low to make any conclusion regarding their specificity.
However, it is possible to conclude that replication of the
lesion-containing plasmids in vitro is relatively accurate.
The percentage of nonhybridizing colonies observed with the replicated
modified DNA was similar to that observed following transformation of
the bacterial tester strain with DpnI-treated, unmodified
replicated plasmid (4 in 240 colonies; 1.6%), supporting the conclusion
that the background is due to the construction and is not related to
the insert modification. In addition, screening of transformants
following direct transformation of the tester strain with these
constructs, without prior incubation in the DNA replication reaction in vitro, also showed that the background of triple inserts,
parent vector, and AatII site mutations was already present in
the construct preparation (data not shown). These results demonstrate
that in vitro replication of plasmids containing either a
single cyclobutane dimer or(6-4) photoproduct at a TT site occurs
without the generation of a large number of mutations targeted to the
lesion site.
These experiments do not address the mechanisms involved in mutagenic and nonmutagenic replication, for example whether gaps are left opposite the lesion or whether replication may be templated by the undamaged strand (see ``Discussion''). The detection of misincorporations as mutations in the transformation assay is dependent on the base changes being present in closed circular or nicked DNA (30) prior to transformation of E. coli MBM7070. It is therefore possible that some misincorporation events, which are present in replicative intermediate DNA forms, for example, would not be detected by this assay.
To investigate the effect of UV-induced DNA damage on DNA
replication and mutagenesis, we have constructed DNA templates
containing a single TT cyclobutane dimer or(6-4) photoproduct and
examined the efficiency with which these constructs are replicated in
human cell extracts in vitro. These two lesions have
substantially different effects on DNA replication in vitro.
Plasmid DNA containing a cyclobutane dimer on the lagging strand
template is replicated as efficiently as an unmodified plasmid in HeLa
cell extracts in vitro. A small reduction in replication
efficiency was observed when the dimer was on the leading strand,
suggesting a potential difference in the effect of this lesion on
leading compared with lagging strand replication. In contrast, the
presence of a(6-4) photoproduct on the lagging strand template inhibits
replication in vitro substantially, by 50%. DpnI-resistant, completely closed circular product DNA from
the replication of the dimer-containing plasmid is sensitive to nicking
by T4 endonuclease V, indicating the presence of a dimer in the product
DNA. It has been suggested that replication of UV-induced lesions may
result in gaps in the newly synthesized strand at the sites of the
lesions, which are subsequently filled in prior to ligation of the
product DNA fragments(32) . While the kinetics of replication in vitro suggest that the steps involved in the replication
reaction are similar in the case of both the undamaged and lagging
strand dimer constructs, a difference could only be observed if the
proposed gap-filling step was the rate-limiting step under the
conditions analyzed here. Given that experiments on the replication of
undamaged SV40-based plasmids in vitro indicate that the
rate-limiting step is initiation at the SV40 origin rather than the
elongation phase (24, 37, 38, 39) ,
it is unlikely that information on the mechanism of DNA synthesis on
the damaged strand could be derived from the kinetics of incorporation
of radioactivity alone. However, analysis of the kinetics of formation
of the products of the DNA replication reaction, by agarose and
polyacrylamide gel electrophoresis, has the potential to provide
important information on the mechanism of replication of damaged DNA in
this system. Recent experiments in which plasmids containing a single
acetylaminofluorene adduct were replicated in vitro indicate
that the undamaged strand may be preferentially replicated in these
templates, providing a potentially error-free mechanism to overcome
lesions in the template(33) . It is possible that a comparable
mechanism could play a role in the inhibition of replication observed
in the presence of the(6-4) photoproduct. Information on the precise
mechanism by which double-stranded DNA containing a UV-induced lesion
is replicated by the complete replication complex will come from a more
detailed analysis of the structure and sequence of nascent DNA formed
during the DNA replication reaction in vitro.
Purified DNA
polymerase I from E. coli has been shown to be capable of
mutagenic bypass of a single cis,syn-cyclobutane pyrimidine
dimer in vitro(34, 35, 36) . The
replication of plasmid DNA containing a single UV-induced lesion,
described here, is consistent with the previous observation that
plasmids containing randomly located UV-induced lesions can be
replicated in mammalian cell extracts in
vitro(17, 18) . Thus, the cell extract contains
the essential cellular proteins involved in this process. Whether
proteins in addition to those known to be necessary and sufficient for
replication of undamaged DNA in vitro(37, 38, 39) are required for
replication of lesion-containing DNA can be elucidated using this
system. In contrast to the present results using a complete replication
system, DNA synthesis by purified DNA polymerase , which is
essential for replication in vitro, is completely blocked by
UV-induced lesions in the template(40) . Another major
replicative DNA polymerase, DNA polymerase
, can carry out
synthesis past a pyrimidine dimer in vitro only in the
presence of the accessory protein proliferating cell nuclear
antigen(41) . These observations suggest that the properties of
the complete replication complex rather than those of the isolated
polymerases may be important in determining the effect of UV-induced
lesions on DNA replication.
The present experiments utilize extracts
from nonirradiated HeLa cells. Extracts from UV-irradiated HeLa cells
have reduced DNA replication activity toward both nonirradiated (10) and randomly UV-irradiated ()pZ189 DNAs in
vitro, suggesting that replication activity following irradiation
is controlled by mechanisms in addition to fork blockage. In mammalian
cells in vivo, UV irradiation results in an arrest of DNA
replication; however, UV-damaged cellular DNA ultimately becomes
replicated, and 24 h after irradiation, newly replicated,
double-stranded DNA contains numerous UV photoproducts in the parental
strand(42) . The observation that, in repair-deficient
mammalian cells from xeroderma pigmentosum patients, UV-damaged DNA is
replicated in the absence of DNA repair (4, 43) is
also consistent with the conclusion that UV-induced lesions do not
constitute an absolute block to DNA replication in vivo. The
differential ability of the two major UV-induced lesions to inhibit DNA
replication may have significance in the cell for the generation of
signals for repair proteins or cell cycle arrest, although the
mechanisms involved in the initial steps of these processes are not
known. The interaction of the DNA replication machinery with RNA
polymerase II transcription complexes, which are stalled at sites of
DNA damage in the template(44, 45) , could also be
important in signaling repair to occur(46) . The recent
demonstration of inducible cellular processes through which replication
can be arrested following exposure to DNA-damaging agents, by, for
example, inhibition of processive DNA synthesis by DNA polymerase
through the action of the p21 protein(16) , suggests that the
replication arrest observed following UV irradiation may be in part
attributable to these processes rather than solely to blockage of
replication fork progression by lesions in the template. In addition,
UV irradiation-induced phosphorylation of replication proteins, such as
human single-stranded DNA-binding protein(10, 47) ,
could play a role in the response of the replication complex to lesions
in the genome of UV-irradiated cells. It is interesting to note
that(6-4) photoproducts are much more efficiently repaired both in
vivo(48, 49, 50) and in cell extracts (51) than are pyrimidine dimers, indicating that this lesion
may be recognized differently by both the replication and the repair
machinery in the cell.
The relative accuracy of replication of the
TT dimer-containing plasmid is consistent with observations from a
number of test systems and from studies of mutations occurring in human
tumors in situ, which indicate that TT sites are
under-represented in the spectrum of UV-induced mutations (reviewed in (4) and (52) ). UV-induced mutations tend to be
predominantly at C-containing lesion sites. It has been proposed that
the A-rule may account for the relative low mutagenicity of lesions at
TT sites, such that the replicative polymerase preferentially inserts
an A opposite a lesion site, leading to the correct base opposite a T
in the lesion. However, evidence from studies on site-specific lesions
in E. coli indicates that different lesions may have different
capacities to code for the correct base, suggesting that the A-rule may
not fully explain the observed lack of mutagenesis in all
cases(19, 20, 21, 22, 23) .
In E. coli, replication of a single-stranded vector containing
a single cis, syn-TT dimer in SOS-induced cells yields 7.6%
targeted mutations(19) . In contrast, the same lesion
replicated in the yeast Saccharomyces cerevisiae yields only
0.4% targeted mutations(22, 23) , indicating that the
mutagenicity of this lesion can differ by almost 20-fold in different
organisms. The(6-4) photoproduct has been shown to be more highly
mutagenic than the cyclobutane dimer in bacteria, yielding 90% targeted
mutations in SOS-induced cells, possibly due to its greater ability to
distort the structure of the DNA backbone(21) . In contrast,
replication of single-stranded vectors containing a single(6-4)
photoproduct in S. cerevisiae results in only 12-20%
targeted mutations, possibly reflecting differences in the ability of
yeast and E. coli DNA polymerases to copy this
lesion(53) . The apparent low mutagenicity of this lesion
following replication in mammalian cell extracts in vitro could also reflect a difference between the responses of the
bacterial and mammalian replication complexes to this lesion. However,
several characteristics of replication in vitro must be
considered in the interpretation of these results. It should be
considered that in vitro, differences in the rate of
elongation of molecules containing a misincorporation at the lesion
site, such that these molecules are not processed into Form I DNA
during the incubation period and so fail to transform the bacterial
tester strain, could also influence the observed mutation frequency. In
addition, in vitro replication of plasmids carrying certain
site-specifically located single acetylaminofluorene adducts has shown
that replication of the most mutagenic acetylaminofluorene adduct is
99% accurate in vitro(33) . In these experiments,
the undamaged DNA strand was found to be preferentially replicated in
the modified construct(33) . In the absence of direct evidence
that the (6-4) lesion has been copied, it is possible that the low
mutagenicity of the(6-4) lesion observed in the present experiments is
a consequence of replication of only the unmodified strand rather than
representing an inherent characteristic of replication of this lesion.
However, while these features of DNA replication in vitro may
lead to a lower estimate of the mutagenicity of certain lesions, the
system also provides a means to directly analyze the relationship
between synthesis arrest, lesion bypass, and mutagenicity. In
particular, direct analysis of the newly replicated nascent DNA around
the lesion site for sequence changes may be possible, providing further
insight into the process of mutation fixation.
The approach described should be useful in defining more completely the molecular basis of replication of UV-damaged templates and in characterizing the mutagenic potential of both UV- and chemical-induced lesions in the mammalian genome.