From the Laboratory of Molecular Genetics, NIEHS,
National Institutes of Health, Research Triangle Park, North Carolina
27709 and the § Department of Chemistry and Center in
Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235
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ABSTRACT |
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We have previously developed an in
vitro system that allows quantitative evaluation of the fidelity
of transcription during synthesis on a natural template in the presence
of all four nucleotides. Here, we have employed this system using a TAA
ochre codon reversion assay to examine the fidelity of transcription by
T7 RNA polymerase past an adenine residue adducted at the
N6-position with ()-anti-trans- or
(+)-anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7
RNAP was capable of transcribing past either BPDE isomer to generate
full-length run-off transcripts. The extent of bypass was found to be
32% for the (
)-anti-trans-isomer and 18% for the
(+)-anti-trans-isomer. Transcription past both adducts was
highly mutagenic. The reversion frequency of bypass synthesis of the
(
)-anti-trans-isomer was elevated 11,000-fold and that of
the (+)-anti-trans-isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the
adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template,
three- and six-base deletions were also observed. These results suggest
that transcriptional errors, particularly with regard to damage bypass,
may contribute to the mutational burden of the cell.
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INTRODUCTION |
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It is well documented that genomic instability can result from errors made during DNA replication, repair, or recombination. However, less is known concerning the effects of inaccurate transcription and/or translation on the integrity of the genetic information. Transcriptional and translational errors may lead to production of mutant proteins. If the defective protein is involved in DNA replication or repair, then even its transient presence may result in permanent changes in the cell's DNA. In fact, a recent paper by Slupska et al. (1) suggests that translational miscoding may result in a mutator phenotype in Escherichia coli due to production of mutant DNA polymerases with dysfunctional proofreading activity.
A likely source of transcriptional errors is damage to the DNA template
and/or ribonucleotide pools. Some DNA lesions, such as UV light-induced
cyclobutane dimers, block the progression of RNA polymerase (2). Such
lesions have been shown to be preferentially repaired, relative to
other parts of the genome, when present in the template strand of an
actively transcribed gene (3-4). However, lesions that are bypassed by
the polymerase may result in erroneous transcripts that, if translated,
will give rise to mutant proteins. The biological consequences of
transcriptional mutagenesis may be particularly significant in
nondividing cells in which some mutant proteins may accumulate over
time. Evidence presented by van Leeuwen et al. (5) points to
transcription errors as a possible source of a mutant form of
-amyloid precursor protein found in neurons of Alzheimer's and
Down's syndrome patients. Deposition of this mutant protein in
neuritic plaque is probably involved in neuron degeneration.
A recent study indicates that oxidative damage to ribonucleotide pools
may result, through transcriptional miscoding, in production of mutant
proteins. Taddei et al. (6) observed an increase in
phenotypic suppression of the Lac phenotype in
MutT
E. coli relative to MutT+ as
a result of 8-oxo-GTP misincorporation during transcription. The
E. coli MutT protein, which is known to degrade 8-oxo-dGTP (7), the product of dGTP oxidation, was shown to also degrade 8-oxo-GTP. The finding that MutT is responsible for cleansing the rNTP
pools raises the question of whether other mechanisms exist, such as
RNA-specific repair processes, to control the production of mutant
proteins.
Our knowledge of how RNA polymerases react to various spontaneous or induced DNA lesions is limited. In particular, little is known about the mutagenic potential of transcriptional damage bypass synthesis. Several recent studies have examined the behavior of SP6 and/or T7 RNAP1 at small, nondistortive DNA lesions such as abasic sites, 8-oxoguanine, and dihydrouracil. These results indicate that all three lesions are easily bypassed and that bypass involves a misincorporation event, most often insertion of an A opposite the lesion (8-10). Unfortunately, the methods used in these studies were not quantitative and only sensitive enough to detect high frequency events.
We have developed an in vitro system that allows
quantitative assessment of the fidelity of
transcription.2 Here, this
system is employed to examine transcription by T7 RNAP past an adenine
residue adducted with ()-anti-trans- or (+)-anti-trans- benzo[a]pyrene diol epoxide (BPDE). BPDE
is a metabolite of the common environmental pollutant,
benzo[a]pyrene. The stereochemistry of BPDE has an influence on its
potent mutagenic and carcinogenic properties (11-14). It has been
shown that BPDE-adducted DNA blocks synthesis by a variety of DNA
polymerases (15-19). Several studies have also demonstrated that BPDE
adducts in DNA impede transcription (20-27). The primary product
formed when BPDE reacts with DNA is deoxyguanosine adducted at the
N2-position. Adenine adducts at the N6-position
are also formed but in smaller amounts (11). Studies have shown that at
high, nonphysiological concentrations, BPDE induces mutations primarily
at dG. However, at lower, more physiological doses, mutations at dA
become much more significant (28-30).
In this study, we demonstrate that T7 RNAP is found capable of transcribing past BPDE-adducted adenine and report on the fidelity of this bypass synthesis.
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EXPERIMENTAL PROCEDURES |
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Materials-- E. coli strains, bacteriophage, and gapped substrate preparation have been described previously (31). RNase H-deficient Moloney murine leukemia virus reverse transcriptase (SuperScript II) and T4 DNA ligase were obtained from Life Technologies, Inc. T7 RNA polymerase was obtained from Promega (Madison, WI), T4 polynucleotide kinase was obtained from New England BioLabs (Beverly, MA), and Sequenase version 2.0 was obtained from Amersham Pharmacia Biotech. AmpliTaq DNA polymerase was purchased from Perkin-Elmer.
Synthesis of Stereochemically Defined N6
Deoxyadenosine Adducts of BPDE--
The two stereospecific BPDE
adducts were constructed on adenine N6 at position 10 within a 16-base deoxyoligonucleotide. The 16-mers, 5'-GTAAAACTTAAGCCAG-3' (lacZ, positions +46 to +75), were
modified by the postoligomerization methodology previously described
(32-34) except that purification was performed on a reversed phase
column (YMC-ODS-AQ (4.6 × 250 mm) using a linear gradient of 100 mM ammonium formate, pH 6.5, containing 9.5-11%
CH3CN over 25 min at a flow rate of 1.25 ml/min; the
elution times for the 10R and the 10S isomers were 17.69 min and 19.40 min, respectively. The collected peaks were lyophilized, redissolved,
and desalted on OPCTM cartridges according to the manufacturer's
directions (Applied Biosystems, Foster City, CA). Peak 1 (10R isomer)
base composition based on enzyme digestion was as follows: dC (3.0); dG
(3.1); T (2.7); dA (5.8); dAN6(10R)-BPDE (1.2); theory: dC
(3.0); dG (3.0); T (3.0); dA (6.0); dAN6(10R)-BPDE (1.0).
Mass spectrometry (electrospray) calculated Mr
was 5200.49; observed ions 1731.9 (M
3H)/3z, 1298.4 (M
4H)/4z, 1039.3 (M
5H)/5z, 865.8 (M
6H)/6z, representing
a measured mass of 5199.66. Peak 2 (10S isomer) enzyme digestion was as
follows: dC (3.0); dG (3.0); T (2.7); dA (5.7);
dAN6(10S)-BPDE (1.2); theory: dC (3.0); dG (3.0); T (3.0);
dA (6.0); dAN6(10S)-BPDE (1.0). Mass spectrometry
(electrospray) calculated Mr was 5200.49; observed ions 1299.2 (M
4H)/4z, 1039.3 (M
5H)/5z, 865.9 (M
6H)/6z, 742.1 (M
7H)/7z, representing a measured
mass of 5201.23. Purity was checked by capillary gel electrophoresis
and denaturing polyacrylamide gel electrophoresis of
32P-end-labeled samples. Each peak contained ~15% of the
other isomer.
Construction of the Ochre Codon-containing Transcription
Template--
The DNA substrate for the ochre codon-based
transcription assay was constructed as follows. Site-directed
mutagenesis was performed (35) using M13mp2SV (36) to replace the ninth
codon of the lacZ gene (GTC, positions +66 to +68, where
position +1 is the first transcribed base) with an ochre (TAA) codon.
In addition, a silent change to the eighth codon was made (GCC
GCT)
that resulted in a unique recognition site for the restriction
endonuclease AflII.
Transcription Reactions--
Transcription reactions (50 µl)
were performed as described previously (37)2 and contained
40 mM Tris-HCl, pH 7.5; 6 mM MgCl2;
2 mM spermidine; 10 mM NaCl; 10 mM
dithiothreitol; 50 units of ribonuclease inhibitor (RNasin, Promega);
200 ng of modified or unmodified, linearized M13mp2 DNA; a 500 µM concentration each of ATP, CTP, GTP, and UTP; 10 µCi
of [-32P]GTP (3000 Ci/mmol); and 15 units of T7 RNA
polymerase. Reaction components were mixed at room temperature and then
incubated at 37 °C for 2 h. Template DNA was digested with
RNase-free DNase (Ambion, 1 unit/µg DNA) for 15 min at 37 °C. The
RNA was purified by phenol extraction and ethanol precipitation and
resuspended in RNase-free water. An aliquot of the transcript was
analyzed on a denaturing 4% polyacrylamide gel and visualized with a
PhosphorImager (Molecular Dynamics STORM 860). The average yield of
transcription products, including full-length and truncated ones, was
200 and 170 ng from (
)-anti-trans- and
(+)-anti-trans-BPDE-adducted templates, respectively.
Transcription with the unadducted template generated an average of 510 ng of full-length transcript.
cDNA Synthesis Reaction Conditions--
RNA-templated DNA
synthesis reactions (50 µl) contained 50 mM Tris-HCl, pH
7.5, 6 mM MgCl2, 50 mM KCl, 10 mM dithiothreitol, 100 µM of each dNTP, 10 µCi of [-32P]dCTP (3000 Ci/mmol), 50 units of
RNasin, 10-50 ng of RNA transcript, 2 pmol of a 21-mer DNA
oligonucleotide primer, and 0.5 pmol of RNase H-deficient Moloney
murine leukemia virus reverse transcriptase. The RNA was heated to
65 °C for 5 min and cooled on ice before adding to the other
reagents. Reactions were incubated at 37 °C for 1 h and then
terminated by the addition of EDTA to 15 mM. The RNA:DNA
hybrids were denatured at 80 °C and cooled on ice, and the RNA was
digested with 50 units of RNase A and 800 units of RNase T1 (Amersham
Pharmacia Biotech) at 37 °C for 1 h. The cDNA was
precipitated with isopropyl alcohol, using glycogen as a carrier, and
resuspended in sterile water. An aliquot of the cDNA was analyzed
by electrophoresis on a 4% denaturing polyacrylamide gel and
visualized with a PhosphorImager. The amount of cDNA synthesis was
estimated by cutting out the cDNA bands from the gel and
determining the radioactivity in a 5-ml scintillation cocktail in an
LS7800 scintillation counter (Beckman).
Hybridization of cDNA Fragment to Gapped M13mp2 DNA--
The
cDNA fragment was hybridized to a double-stranded circular M13mp2
substrate that contained a 442-base single-stranded gap. The gap
spanned the sequence in the lacZ gene complementary to
the cDNA (lac positions
216 to +195). A molar excess
of cDNA was mixed with gapped M13mp2 DNA in 300 mM
NaCl, 30 mM sodium citrate, heated to 70 °C, and slowly
cooled to room temperature. The annealed product was analyzed by
electrophoresis in an 0.8% agarose gel and transfected into an
E. coli
-complementation host, and mutations were scored,
all as described previously (31).
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RESULTS |
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Construction of Transcription Templates--
Templates for
transcription were constructed that contained the lacZ
mutational target and the T7 RNA polymerase promoter sequence as
described under "Experimental Procedures." The target for measuring
transcriptional errors, a TAA stop codon, was engineered at positions
+66 to +68 in the lacZ
sequence on the nontranscribed (+)-strand. Opposite the T of the ochre codon, the transcribed strand
contained either
(+)-anti-trans-N6-BPDE-adducted
adenine,
(
)-anti-trans-N6-BPDE-adducted
adenine, or nonadducted adenine (Fig.
1).
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T7 RNA Polymerase Transcription from Templates with a BPDE-adducted
or Unadducted Adenine--
Run-off transcription from
FspI-linearized templates is predicted to generate a
transcript 313 nucleotides in length (from position 118 through
+195). Reactions performed using either an unadducted template or a
template in which adenine at position +66 was adducted with
(+)-anti-trans- or (
)-anti-trans-BPDE generated 313-nucleotide transcripts, as shown in Fig.
2A. In each reaction with an
adducted template, several shorter bands were observed, including one
major band. These truncated transcripts indicate that the polymerase
had stalled or dissociated. We have estimated that the major band
representing the truncated product is approximately 3 nucleotides
shorter than a transcript generated from an AflII-linearized template, which is predicted to yield a 186-nucleotide transcript. If
transcription was aborted after insertion of a nucleotide directly opposite the adduct, the RNA would be 184 nucleotides in length. Therefore, we deduce that the transcript is probably truncated one base
5' of the adduct, at position +65. Based on the amount of full-length
transcript relative to the total amount of products in the lane, there
was 32% bypass for (
)-anti-trans-BPDE and an 18% bypass
for (+)-anti-trans-BPDE (Fig. 2B). The amount of full-length transcript in reactions with the
(
)-anti-trans-BPDE-adducted template was significantly
greater than in reactions with the (+)-anti-trans-BPDE-adducted template. This was verified by
the Wilcoxon rank sum test, which gave a p value of
0.05.
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Fidelity of Transcription Past a BPDE Adduct--
The observation
that T7 RNAP was capable of transcription past the BPDE adducts allowed
us to examine the fidelity of bypass synthesis. Transcriptional
fidelity measurements were obtained using an ochre codon reversion
assay based on complementation of -galactosidase activity by the
-peptide portion of the enzyme. This assay is a modification of a
method that we used previously to examine the fidelity of reverse
transcription (37). Briefly, transcription was carried out from the T7
promoter, which was located 5' to the lacZ
gene. The
run-off transcript was primed with a DNA oligonucleotide and cDNA
synthesis performed. The cDNA was hybridized to a double-stranded,
circular M13mp2 substrate containing a single-stranded gap that spanned
the lacZ
gene complementary to the cDNA. The products
of cDNA hybridization reactions were transfected into an E. coli
-complementation host, which yielded rescued errors as
mutant plaques. This assay focuses specifically on single-base
substitution errors at the three-base mutational target, the TAA ochre
codon in the lacZ
sequence. Accurate transcription of the
ochre codon-containing template results in a white plaque. Of the nine
possible single base substitutions at the TAA codon, seven are
predicted to restore partial or complete lacZ
complementation and result in a blue plaque phenotype, while two result
in nonsense codons and do not yield a phenotypically detectable change
in plaque color (38).
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DISCUSSION |
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The data presented here demonstrate that transcriptional bypass of
a BPDE-adducted template is highly mutagenic. Misincorporations occurred at a rate of 1 in 3 errors per nucleotide incorporated for the
()-anti-trans-isomer and 1 in 6 for the
(+)-anti-trans-isomer. This study is the first quantitative
analysis of the fidelity of site-specific damage bypass synthesis
during transcription.
Although transcription by T7 RNAP was stalled by the
()-anti-trans- and (+)-anti-trans-BPDE-adducted
adenine, generation of full-length transcripts in reactions with the
adducted templates indicated that the polymerase was capable of
synthesizing past both adducts (Fig. 2A). However, the
amount of bypass synthesis appears to depend on the stereochemistry of
the adduct. The observation that on average 40% less full-length
transcript is produced from (+)-anti-trans-BPDE adenine
templates, relative to (
)-anti-trans-BPDE adenine
templates suggests that the (+)-BPDE adenine adduct is more inhibitory
to transcription than the (
)-BPDE adduct. This result is consistent
with the report that the stereochemistry of BPDE-guanine adducts
affects the degree of inhibition of T7 RNAP-dependent
transcription (26).
According to NMR studies of duplex oligodeoxyribonucleotides
containing site-specific BPDE-adducted adenine, the pyrenyl moiety of
the ()-anti-trans-isomer is directed toward the 5'-end of the modified strand (40-42). The (+)-anti-trans-adduct is
oriented mainly toward the 3'-end of the adducted strand, although it
may exist in more than one conformation. A 3'-oriented adduct may sterically hinder an RNA polymerase moving in a 5' to 3' direction and
thus would be more difficult to bypass. Our observation that the
(+)-anti-trans-BPDE adenine adduct was a stronger block to T7 RNAP than the (
)-anti-trans-BPDE adduct is therefore
consistent with this hypothesis. However, the actual conformation of
these adducts at single-strand template-duplex junctions may be
different from that in duplex oligodeoxyribonucleotides.
Full-length transcripts generated with the adducted templates contained
a high level of misincorporation events. The reversion frequency of the
TAA codon was elevated 11,000- and 6000-fold due to transcription
errors on the ()-anti-trans- and
(+)-anti-trans-BPDE-adducted templates, respectively,
relative to reversion frequency in the reaction with the unadducted
template. Thus, it appears that T7 RNAP synthesis past the
(
)-anti-trans-BPDE-adducted adenine was almost twice as
mutagenic as synthesis past the (+)-anti-trans-adduct. How
the efficiency of BPDE bypass and the miscoding potential of the
lesion's stereochemistry are related is an intriguing question.
The observed increase in reversion frequency was due mainly to base
substitution errors. The most frequently recovered errors from
reactions with both adducted templates were T to A transversions followed by T to G transversions. This reversion specificity is consistent with misinsertions opposite the adducted adenine and indicates that T7 RNAP most readily misincorporates A opposite either
of the adducts. Interestingly, adenine was also the most commonly
inserted base opposite an abasic site by T7, SP6, and E. coli RNA polymerases (8, 9). The second most frequently inserted
nucleotide was G. Guanine appeared to be incorporated 1.5- and 9-fold
less frequently, relative to adenine, opposite ()-anti-trans-BPDE and
(+)-anti-trans-BPDE-adducted adenine, respectively. The fact
that we see different proportions of adenine and guanine opposite the
two adducts would argue that the stereochemistry of the adduct plays an
important role in determining the nature of the misinserted
nucleotide.
It has been shown that the stability of a
(+)-anti-trans-BPDE-adducted adenine mismatched with dG is
significantly higher than the stability of the adducted adenine paired
with the correct dT (41, 43). This suggests that the differences in
stability between the incorrect and correct base pairs containing the
different diastereomers may play a role in BPDE-induced mutagenesis.
The difference in the proportion of errors resulting from G
misincorporation relative to A misincorporation opposite the two
adducts may also reflect inequality in the efficiency of extension of
mispairs containing the (+)-anti-trans- or the
()-anti-trans-adducted adenine.
In contrast to the results presented here, bypass of BPDE-adducted guanine by T7 RNAP was reported to be accurate (26). Misinsertions opposite the adducted guanine, mainly of A and to a lesser extent of G, resulted in termination and a truncated transcript. The difference in mutagenicity of bypass between adducted adenine and adducted guanine may be due to the difference in orientation of the adduct within the DNA.
When adducted to guanine, the BPDE moiety lies in the minor groove,
either in a 3' or 5' orientation, depending on the diastereomer (41).
When adducted to adenine, BPDE is partially intercalated in the major
groove, 5' for ()-isomers and 3' for (+)-isomers (40-42). The steric
interaction between the hydroxyls of the pyrene moiety and DNA
nucleotides is reduced when the adduct is intercalated in the major
groove, as it is for adenine adducts. In contrast, guanine adducts lie
in the minor groove (41). The adduct-directed DNA distortion may
stabilize an A:A or A:G mispair, thereby increasing significantly the
probability of a misinsertion event. In addition, normal base pairing
between the adducted adenine and the correct nucleotide may be
destabilized. As mentioned above, the thermostability of a
(+)-anti-trans-BPDE-adenine:guanine mismatch is higher than that of a correct base pair (41), indicating that the adduct alters
even normal base pair stability. In the case of an adducted guanine
template, insertion of the correct nucleotide may be the only means by
which T7 RNAP can accommodate the lesion and synthesize past the
adduct. On the other hand, it is possible that some mutagenic bypass of
the BPDE-adducted guanine takes place. However, this bypass may not
have been detected due to the relatively low sensitivity of the method
used for detection of misincorporations (26).
We observed a significant increase in three- and six-base deletions spanning the adducted site, relative to reactions with the nonadducted template. These results suggest that adenine-adducted BPDE may induce not only base misincorporations but also small deletion mutations during transcription by T7 RNAP. However, in order to evaluate the level and specificity of these mutations, a substrate designed to detect deletion mutants should be used.
In summary, we have shown that BPDE-adenine adducts in the template strand can be bypassed by T7 RNAP and that the bypass is highly mutagenic. Misinsertion of adenine and to a lesser extent of guanine opposite the adduct occurred at a very high frequency. These results suggest that if bypass transcription of unrepaired BPDE-adducted adenine does occur in vivo, it may result in transcripts that contain missense mutations and, hence, code for mutant proteins. Such proteins may not only have cytotoxic effects; by interfering with DNA metabolism, they could alter the stability of genetic information. The potential biological consequences of transcriptional mutagenesis in higher organisms merits further investigation.
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ACKNOWLEDGEMENTS |
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We thank Miriam Sander and Roel M. Schaaper for helpful discussions and for critical evaluation of the manuscript. We gratefully acknowledge the help of Pamela Horton (Vanderbilt University) in the synthesis and purification of the BP-adducted oligonucleotides.
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FOOTNOTES |
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* This work was supported by U.S. Public Health Service Grants ES 00267 and ES 03755.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: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-3535; Fax: 919-541-7613; E-mail: bebenek{at}niehs.nih.gov.
1 The abbreviations used are: BPDE, benzo[a]pyrene-7,8-dihydro-9,10-diol epoxides; T7 RNAP, T7 RNA polymerase; RF, replicative form.
2 K. M. Remington and K. Bebenek, manuscript in preparation.
3 K. M. Remington, S. E. Bennett, C. M. Harris, T. M. Harris, and K. Bebenek, unpublished observations.
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REFERENCES |
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