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(Received for publication, June 27,
1995; and in revised form, September 9, 1995) From the
The model that transcription-coupled excision repair reflects
the interference of DNA damage with the transcription process predicts
that the rate of such excision repair will be related to the degree to
which a particular type of lesion blocks transcription. We tested this
by measuring the rate of excision repair of guanine adducts formed in
the HPRT gene of diploid human fibroblasts and in the overall
genome by two structurally related polycyclic carcinogens,
1-nitrosopyrene (1-NOP) and N-acetoxy-2-acetylaminofluorene
(N-AcO-AAF) and comparing the results with those we found previously
using benzo[a]pyrene diol epoxide (BPDE). We also measured
the degree of interference with in vitro transcription by
these adducts. Our results showed that, although BPDE adducts are four
times more effective than 1-NOP adducts in blocking transcription, the
preferential and strand-specific repair of 1-NOP adducts was twice as
fast as that of BPDE adducts. Excision repair of N-AcO-AAF adducts was
significantly slower than that of BPDE adducts and was not
strand-specific. The efficiency of blocking of transcription by
deacetylated N-AcO-AAF adducts was similar to 1-NOP adducts. Therefore,
the extent to which a particular lesion blocks transcription in
vitro does not predict its rate of preferential or
transcription-coupled excision repair.
Ultraviolet light-induced cyclobutane pyrimidine dimers (CPD) ( Such
strand-specific repair, which affects the spectrum of mutations in
active genes(4, 5) , has been shown to be the result
of coupling of transcription and nucleotide excision repair. In Escherichia coli, transcription and repair are coupled by the mfd gene product(6) , and mutations in the human
homolog have been postulated to be the genetic defect in some patients
with Cockayne syndrome, a disorder of development and DNA
repair(7, 8) . To explain this preferential and
transcription-coupled repair, models have been proposed in which RNA
polymerase that is stalled at a CPD acts as a signal to the mfd-like gene product, which releases the stalled RNA
polymerase, or causes it to back up, thus exposing the damage and
targeting the excision repair apparatus to the
lesion(9, 10) . This model predicts that the rate of
preferential excision repair of a specific type of DNA lesion in the
transcribed strand of an active gene will be proportional to the extent
to which the lesion blocks transcription. To test this hypothesis, and
to determine whether structurally related polycyclic adducts are
preferentially repaired and exhibit strand-specific repair in diploid
human fibroblasts in culture, we measured the rate of repair of adducts
formed by 1-nitrosopyrene (1-NOP) and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) from the
individual strands of the HPRT gene and from the overall
genome, and compared the results with those we had obtained with
BPDE-induced adducts. We also compared the degree of interference with
T7 RNA polymerase transcription caused by adducts formed by these
carcinogens. All three agents bind principally to guanine. BPDE, a
reactive intermediate of benzo[a]pyrene, binds to the
N
Figure 1:
Structures of the adducts induced by
N-AcO-AAF and by 1-NOP.
dG-C8-AP was much less effective than dG-N
Purified DNA (12 µg) was used for repair analysis. As an
internal standard, 10 pg of the plasmid used to synthesize the
riboprobe was added to each sample. The samples were divided in half;
one half was exposed to UvrABC excinuclease (17 pmol of each subunit);
the other to excinuclease buffer alone. The enzyme subunits were a
generous gift from Dr. P. van de Putte (Leiden University, The
Netherlands). After 1 h at 37 °C, the reaction was stopped by the
addition of proteinase K and SDS, and the DNA was purified by
ultrafiltration and precipitated with EtOH. The DNA samples were
denatured in 90% formamide and analyzed by electrophoresis in Tris
borate/EDTA buffer as described previously(3) . After
electrophoresis, the gel was stained with ethidium bromide,
acid-depurinated, equilibrated with 0.4 N NaOH, and
transferred for 40-48 h to a Zeta Probe GT membrane (Bio-Rad)
under alkaline conditions(3) . Plasmid pG2pa(24) ,
constructed by subcloning a 1.4 kb of EcoRI-XhoI
fragment containing sequences from intron I of the human HPRT gene into the vector pGEM2, was kindly provided by A. C. Chinault
(Baylor College, Houston, TX). It contains the SP6 and T7 promoters on
either side of the insert. RNA transcripts hybridizing
strand-specifically were generated as described
previously(3, 25) . Hybridizations were performed in 5
ml of solution as described previously(3) , using The intensities of the bands were
quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
The intensity of the full-length fragment band was normalized with the
internal standard band. The average number of UvrABC-sensitive
sites/fragment was calculated by the Poisson distribution(26) .
These calculations took into consideration the very low number of
nonspecific incisions produced by the UvrABC excinuclease.
Unbound carcinogen was removed by phenol-chloroform extraction
followed by four successive ethanol precipitations. The mol of The plasmid was digested with EcoRI followed by phenol-chloroform extraction and ethanol
precipitation. Completion of digestion was confirmed by electrophoresis
in an agarose minigel. Plasmid DNA (600 ng) with an adduct level of
0-10.5 dG-C8-AP, 0-9.6 dG-C8-AF, or 0-2.6 BPDE
adducts/transcribed strand was used as a template in the transcription
reaction as described previously (3) except that the reaction
was terminated after 15 min by passing the reaction mixture through a
Sephadex G-50 spin column to remove unincorporated nucleotides. A
5-µl aliquot was taken for determination of incorporation of
[
As expected, in the
control populations DNA replication began
Figure 2:
Autoradiograms illustrating the extent of
repair of dG-C8-AP in the transcribed (T) and the
nontranscribed (NT) strand of the HPRT gene. DNA was
isolated from untreated human cells (first two lanes) or from
cells with the indicated incubation period (H) following
treatment with 0.3 µM 1-NOP (C). DNA samples were
digested with BamHI, and plasmid pG2pa containing the
sequences to be probed was added as an internal control. The samples
were then treated (+) or not treated(-) with UvrABC (E) and subjected to electrophoresis and Southern
hybridization with
Figure 3:
Autoradiograms illustrating the extent of
repair of dG-C8-AF in the transcribed (T) and the
nontranscribed (NT) strand of the HPRT gene. Cells
were incubated for the indicated number of hours (H) following
treatment with 1.2 µM N-AcO-AAF (C). Analysis of
UvrABC-sensitive (E) sites as in Fig. 2.
The results are shown in Table 2and are compared with each
other and with the rates for overall genome in Fig. 4. In each
instance, repair of adducts induced by 1-NOP was rapid and
strand-specific; 80% of the dG-C8-AP in the transcribed strand had been
removed in 8 h, and repair was virtually complete in 16-20 h.
Repair in the nontranscribed strand was slower but was also close to
completion (85%) by 16-20 h. The greatest difference in repair of
the two strands was found after 4 h, when repair of the transcribed
strand was 60-64% complete and of the nontranscribed strand was
30-31% complete. This 2-fold difference is equal to, or greater
than, that of CPD in the same gene(2) . Although this
difference is less striking than in rodent cells, which have minimal
repair of the nontranscribed strand, CPD in active genes of human cells
are widely recognized to be strand-specifically repaired. In addition,
the rate of repair of dG-C8-AP adducts in the HPRT gene was
faster than that in the overall genome. In contrast, little if any
repair of dG-C8-AF was detected after 5 h of repair. After 15 h, only
33% of these adducts had been removed, and the rate of repair of
adducts in the transcribed strand was not faster than in the
nontranscribed strand. This rate was only slightly faster than the rate
of repair from the genome overall during the same time period.
Figure 4:
Rate of removal of 1-NOP-induced adducts (squares) and N-AcO-AAF-induced adducts (circles)
from the transcribed (closed symbols) and the nontranscribed (open symbols) strand of the HPRT gene as determined
using UvrABC excinuclease, and from the genome overall (half-filled
symbols, dashed lines) as determined from loss of
tritiated carcinogen from parental DNA. With the exception of the rate
of repair of N-AcO-AAF adducts from the overall genome, cells were
synchronized, treated in early G
Figure 5:
The number of adducts per plasmid was
determined by reacting 1-[
The basis of the use of Southern blotting as a method to
determine rates of repair in specific sequences is the ability of the
fragment of interest to remain full-size, i.e. the probability
that the fragment of interest does not contain any damage that would
cause it to be cut into a smaller sized fragment. If one can assume
that such damage is randomly distributed among the population of HPRT genes and is sensitive to enzymatic excision, the Poisson
distribution function, i.e. P(0) = e We determined the rate of
nucleotide excision repair of adducts induced in DNA by the carcinogens
1-NOP and N-AcO-AAF in the individual strands of the HPRT gene
and in the overall genome (global repair). Our results indicate that
dG-C8-AP is repaired rapidly and that the excision repair is
preferential and strand-specific and twice as rapid as the rate of
excision of corresponding dG-N Comparison of the
structure of these adducts (Fig. 1) reveals that they are quite
similar, although dG-C8-AP has four aromatic rings, whereas dG-C8-AF
has two aromatic rings connected by a cyclopentane ring. NMR analysis
of the conformation adopted by duplex DNA containing dG-C8-AF shows
that the adduct does not cause substantial helix perturbation (34) . The adducted guanine remains in the normal anti conformation and is available for base pairing(35) . The
adduct preferentially resides in the major groove of a relatively
undisturbed B-type DNA(34, 35) . More recently, Cho et al.(18) reported that the planar arylamine has
minor conformers that may represent stacking interactions with
neighboring bases, which could cause significant local conformational
perturbation. Much less information on the conformation adopted by DNA
containing dG-C8-AP is available, but Nolan et al.(17) showed that such adducts also lie in the major groove
of DNA and cause little helical distortion but that there may be
significant stacking interactions with neighboring bases. Despite
the apparent structural similarity of the two kinds of adducts,
previous studies from this laboratory have shown marked differences in
their cytotoxic and mutagenic effects. In a shuttle vector system, in
which dG-C8-AP (23) or dG-C8-AF(36) were formed in plasmid
pZ189 in vitro and the plasmid was allowed to replicate in
repair-proficient human cells and then analyzed for mutations induced
in a bacterial target gene carried on the plasmid, dG-C8-AP was found
to be 4 times as effective in interfering with bacterial transformation
than dG-C8-AF. Nevertheless, the frequency of mutations induced in the supF gene during replication of the plasmid in human cells was
similar, as were the kinds of mutations seen. The location of base
substitutions in the target gene (spectrum) was not
identical(23, 36) . Earlier studies in this laboratory
on the cytotoxic and mutagenic effect of dG-C8-AP and dG-C8-AF in
repair-proficient human cells showed that a level of 25
dG-C8-AP/10 The molecular basis for the
observed differences in cytotoxicity, mutagenicity, and rates of repair
of structurally-related adducts may well lie in the local
conformational alterations induced by DNA-adduct formation. Although
limited data exist on the structure of dG-C8-AP and dG-C8-AF, general
structural features that influence the biological response to these
adducts can be inferred by comparison to the extensively studied BPDE
adducts. In contrast to dG-C8-AP or dG-C8-AF, BPDE adducts form
primarily at the N Sancar and colleagues (40) have recently proposed a model to explain the substrate
specificity of the human repair excinuclease system in which the
recognition subunit forms a stable complex with DNA in the region of
adducted bases. Formation of the stable complex includes melting of the
DNA in the region of the adduct. Such local unwinding is facilitated by
adducts that interfere with base pairing, such as the N
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 27222-27227
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)are excised more rapidly from transcriptionally active
regions than from inactive regions of the genome of human cells
(preferential repair) (1) and more rapidly from the transcribed
strand of active genes than from the complementary nontranscribed
strand (strand-specific repair). Synchronized populations of diploid
human fibroblasts irradiated with 10 J/m
excise 50% of the
CPD in the transcribed strand of the hypoxanthine
phosphoribosyltransferase (HPRT) gene within 4 h and in the
nontranscribed strand within 8 h; 50% are excised from the overall
genome in 12 h(2) . In a similarly designed study, Chen et al.(3) showed that excision repair of
benzo[a]pyrene diol epoxide (BPDE)-induced adducts is also
preferential and strand-specific. However, the rate of excision of BPDE
adducts by these cells is 2-fold slower than that of CPD.
position of guanine by a covalent bond between the
nitrogen and the carbon at position 10 where the epoxide was located
(dG-N-BP)(11) . The adduct is pentacyclic and lies
in the minor groove of the DNA molecule(12) . 1-NOP is a
partially reduced metabolite of 1-nitropyrene, a major environmental
pollutant and a by-product of incomplete diesel
combustion(13) . 1-NOP must undergo a further intracellular
reduction step before it forms an unstable reactive intermediate that
binds covalently to the C-8 position of guanine to form the stable
tetracyclic adduct N-(deoxyguanosin-8-yl)-1-aminopyrene
(dG-C8-AP) (14) (Fig. 1). N-AcO-AAF is a direct acting
compound that spontaneously loses the acetoxy ester, generating a
reactive electrophilic intermediate that also binds principally
(>95%) to the C-8 position of guanine(15) . In human cells,
N-AcO-AAF is rapidly deacetylated before binding so that its principal
adduct is the tricyclic deacetylated AF residue on the C-8 position of
guanine, i.e. N-(deoxyguanosin-8-yl)-2-aminofluorene
(dG-C8-AF) (16) (Fig. 1). Both dG-C8-AP and dG-C8-AF
lie in the major groove of the DNA molecule(17, 18) .
-BP in
blocking T7 RNA polymerase transcription. Repair of dG-C8-AP was
preferential, strand-specific, and very rapid, i.e. twice as
fast as repair of dG-N-BP. In contrast dG-C8-AF, which
blocked transcription to the same extent as dG-C8-AP, was repaired
slowly, and the repair was not strand-specific. The results indicate
that the extent to which an adduct arrests transcription in vitro does not predict its rate of transcription-coupled repair.
Cell Culture and Synchronization
Diploid human
fibroblasts from the foreskin of a neonate (19) were cultured
in Eagle's minimal essential medium modified as described
previously (20) containing 10% supplemented calf serum
(Hyclone, Logan, UT). Cells were driven into G
by density
inhibition and nutrient deprivation as described
previously(21) . To allow the cells to re-enter the cell cycle,
they were released from confluence and replated at a density of
10
cells/cm. Incorporation of
[
H]thymidine into acid-insoluble material
indicated that under these conditions the time of onset of S-phase
after release from G was approximately 16 h.Determination of the Initial Number of 1-NOP- or
N-AcO-AAFInduced DNA Adducts in Genomic DNA and Their Rate of Removal
from the Overall Genome
To determine the rate of excision from
the overall genome, we measured loss of radiolabeled adducts as a
function of time posttreatment. Generally tritiated 1-NOP (specific
activity, 177 mCi/mmol) was a generous gift from Dr. Frederick Beland,
National Center for Toxicological Research (Jefferson, AR). The
compound was dissolved in anhydrous dimethyl sulfoxide
(MeSO) and stored at -20 °C under N until just prior to use. Generally tritiated N-AcO-AAF (specific
activity, 484 mCi/mmol) was obtained from Chemsyn Science Laboratories
(Lenexa, KS) dissolved in tetrahydrofuran. The culture medium on the
cells was removed, the cells were rinsed with phosphate-buffered
saline, and the medium was replaced with serum-free medium. The
compound diluted in MeSO was delivered into the individual
dishes by micropipette. Preliminary studies showed that, unlike what
happens with N-AcO-AAF adducts, with 1-NOP-induced damage at a level of
about 0.5 adducts/20 kb (25/10
nucleotides), the onset of
S-phase was delayed long enough to allow measurements over a 16-h
period. Therefore, for repair of 1-NOP adducts from the overall genome,
the cells were synchronized and treated for 1 h in early G phase. Immediately following treatment or after 5 or 16 h of
incubation in fresh culture medium, the cells were harvested, and the
DNA was isolated as described previously(22) . The number of
covalently bound residues (adducts) per 10 nucleotides was
determined from the A of the DNA solution and
the specific radioactivity. To avoid possible interference with repair
studies as the result of S-phase DNA replication, for N-AcO-AAF a
double-label technique was used. The DNA in asynchronously growing
cells was labeled with [
C]thymidine (specific
activity, 59 mCi/mmol) for 48 h. Then, the cells were exposed to
tritiated N-AcO-AAF for 30 min and harvested immediately or after 18,
40, or 72 h of incubation in fresh culture medium, and the DNA was
isolated as described previously(22) . The initial number of AF
residues bound was determined as above, and the percentage remaining on
parental DNA at the indicated times was calculated from the H/C ratio at these times divided by the ratio
at time zero, as described previously(23) .Determining Rates of Repair in the Individual Strands of
the HPRT Gene
The methods used are essentially those we employed
previously to measure strand-specific repair of BPDE adducts in human
fibroblasts(3) . Briefly, synchronized populations of cells
were treated with 1-NOP or N-AcO-AAF for 1 h in early
G-phase and harvested immediately or after the indicated
times for repair. The cells were washed with phosphate-buffered saline
and lysed in lysis buffer. The cell lysates were incubated at 56 °C
for 8-12 h. DNA was extracted as described
previously(22) , precipitated, resuspended in TE buffer, and
treated with RNase A. The DNA was re-extracted as above, precipitated
with EtOH, and dissolved in TE buffer at 0.2 mg/ml. The purified DNA
was digested with BamHI (5 units/µg DNA) overnight at 37
°C; the completion of digestion was verified by electrophoresis,
and the digested DNA was purified by extraction and precipitation as
above.10
cpm of P-labeled probe at 42 °C for 20-24
h. After hybridization, the membranes were washed as described and
exposed to Kodak XAR-5 x-ray films with intensifying screens or to
phosphor storage cassettes.
Determining the Degree of Interference With RNA
Transcription Induced by dG-C8-AP, dG-C8-AF, or
dG-N
For dG-C8-AP, plasmid pG2pa was
dissolved in helium-purged sodium citrate buffer (10 mM, pH
5.0) at a concentration of 250 µg/ml. A 5-µl aliquot of a
freshly prepared solution of ascorbic acid (20 mM in
H-BP0) was added to provide needed reduction of 1-NOP,
followed by 0-15 µl of a 0.5 mM stock solution of
generally tritiated 1-NOP dissolved in MeSO. The final
reaction volume was 200 µl. The samples were mixed and incubated
for 3 h at 37 °C. For dG-C8-AF, plasmid pG2pa was treated with
generally tritiated N-hydroxy-2-aminofluorene exactly as
described for 1-NOP, except ascorbic acid was omitted. The stock
concentration was 1.4 mM, and the compound was dissolved in
anhydrous ethanol. For BPDE, plasmid pG2pa was dissolved in 10 mM Tris, 1 mM EDTA, pH 8.0, at a concentration of 500
µg/ml. Aliquots (0-12 µl) of 0.7 mM [H]BPDE (specific activity, 0.885 Ci/mmol),
dissolved in tetrahydrofuran, were added to 200 µl of the plasmid
solution. The samples were mixed and incubated at room temperature for
2 h.H-labeled adduct bound per mol of plasmid was calculated
from the A of the DNA and the specific activity,
and the average number of adducts per transcribed strand of the 1.4-kb HPRT fragment was estimated.
-P]UTP into trichloroacetic
acid-precipitable counts.
Determining the Delay in Onset of DNA Replication
Induced by Carcinogen Treatment
The rate of repair of carcinogen
adducts from DNA is determined from the frequency of adducts initially
formed in DNA and that remaining in DNA after particular periods of
time. Two methods are used, one measures the frequency of UvrABC
excinuclease-sensitive sites in the 5`-half of the HPRT gene
and the other measures the frequency of radiolabeled adducts in total
DNA. With either method, S-phase replication of DNA by the cells
following treatment with carcinogen will dilute pre-existing parental
DNA and give the appearance of a decrease in the frequency of adducts.
For the majority of the experiments to be discussed, S-phase DNA
replication did not occur because the target population was
synchronized and exposed to carcinogen in early G-phase,
and the rate of repair was determined during the period prior to the
onset of S-phase DNA synthesis. Watanabe et al.(21) in this laboratory showed that diploid human
fibroblasts synchronized by being released from the density-inhibited
G-state by being plated at 10 cells/cm begin DNA replication after 16 h. Treatment with polycyclic
aromatic carcinogens causes a delay in the onset of such replication.
To determine the extent of delay caused by the concentrations of 1-NOP
or N-AcO-AAF to be used in the majority of the DNA repair studies, i.e. concentrations designed to introduce approximately one
adduct/20-kb fragment of the HPRT gene (50 adducts/10
nucleotides), we released cells from the densityinhibited resting
state by plating them into a series of dishes at 10 cells/cm, treated them 5 h later (in early
G-phase) with 1-NOP or N-AcO-AAF, and assayed the
incorporation of [H]thymidine into acid-insoluble
material at various times after treatment.16 h after release.
Cells treated with 0.3 µM 1-NOP showed no incorporation of
[
H]thymidine whatsoever during the 20-h period
examined posttreatment. Cells treated with 1.2 µM N-AcO-AAF began DNA replication approximately 19 h after
treatment, i.e. 24 h after release. This is at least an 8-h
delay in the scheduled onset of S-phase. The absence of DNA replication
during the period of interest, i.e. up to 19 h after exposure
to N-AcO-AAF and up to at least 20 h after exposure to 1-NOP,
simplified most of the assays of repair reported here because there was
no dilution of parental DNA with nascent DNA during those times.Determining the Rate of Repair of dG-C8-AP and dG-C8-AF
from the Overall Genome
Synchronized cells were exposed to 0.5
µMH-labeled 1-NOP in early
G-phase. The cells exhibited a survival of 30% and an
initial adduct frequency of 27/10 nucleotides. The rate of
repair is shown in Table 1. In the case of repair of N-AcO-AAF
adducts in the overall genome, a double label technique was used to
control for DNA replication. Asynchronously-growing cells, prelabeled
with [C]thymidine to be able to distinguish
preexisting parental DNA, were treated with 0.6 µM or 1.6
µMH-labeled N-AcO-AAF. These cells exhibited
an initial adduct frequency of 18 or 38/10 nucleotides,
respectively. Their slower rate of repair is also shown in Table 1. This difference in rate did not reflect a greater
cytotoxic effect (Table 1).
Evidence of Strand-Specific Repair of dG-C8-AP But Not
dG-C8-AF
Synchronized populations of cells were treated with 0.3
µM 1-NOP or 1.2 µM N-AcO-AAF in early
G-phase (5 h post release from G). The UvrABC
excinuclease-sensitive sites initially induced in the individual
strands of the 20-kb BamHI fragment of the human HPRT gene or remaining after various lengths of time for repair were
determined using Southern blotting(3) . This fragment, which is
in the 5`-portion of the gene, is entirely within the transcription
unit(3) . To avoid detection of pseudogenes, it was probed with
a 1.4-kb strand-specific riboprobe that anneals to an intron
sequence(27) . Representative autoradiograms of such repair
studies are presented in Fig. 2and Fig. 3. The
intensities of the full-length (20 kb) fragments were determined by
densitometric scanning of the autoradiograms and/or by analysis of the
image left on storage phosphor cassettes by the blots using a Molecular
Dynamics PhosphorImager. The two methods yielded equivalent results.
P-labeled probes. For clarity, the
bands corresponding to the internal controls have been
omitted.
-phase, and examined for
repair prior to the onset of S-phase. With the former, cells were
prelabeled with [C]thymidine, exposed to
tritiated N-AcO-AAF in exponential growth, and examined for the ratio
of H to C in purified DNA to determine the
rate of loss of dG-C8-AF.
Interference of dG-C8-AP, dG-C8-AF, or
dG-N
If the degree of
interference with transcription is a predictor of the rate at which a
particular type of DNA damage is excised from active genes, then
dG-C8-AP ought to be an even more effective block to transcription than
dG-N-BP with RNA Transcription-BP. dG-C8-AF ought to block transcription minimally,
if at all. To test this, we treated plasmid pG2pa with various
concentrations of tritiated 1-NOP, N-OH-AF, or BPDE and determined the
number of adducts per plasmid and the degree of interference with the
extent of incorporation of P-labeled UTP into nascent
transcripts. The results (Fig. 5) showed that the prediction
does not hold true. It required an average of 6.8 to 7.0 dG-C8-AP or
dG-C8-AF in the transcribed strand to reduce transcription to 37% of
the untreated control, whereas with dG-N
-BP, 1.6 was
sufficient.
H]NOP with pG2pa under
acidic and reducing conditions, H-labeled N-OH-AF under
acidic conditions, or [H]BPDE under standard
conditions, and removing unbound H-labeled carcinogen by
phenol chloroform extraction followed by four successive ethanol
precipitations. The molar ratio of adducts to plasmid was determined by
the A of the DNA and specific activity of the
H-labeled carcinogen. The average number of adducts per
transcribed strand was estimated based on the size of the insert
relative to the plasmid. Inhibition of transcription was determined by
digesting the plasmids with EcoRI, using them as a template in
transcription reactions and determining the fmol of
[-P]UTP incorporated into acid-precipitable
material. The incorporation into the transcripts directed by the
control template (no adducts) was set at 100%. BP, adducts
induced by BPDE (
-
-); AP, adducts induced by 1-NOP (
- -
-); AF, adducts induced by N-OH-AF
(-
).
, where P(0) is the
percentage of fragments with no damage and x is the average
number of enzyme- sensitive sites/fragment, yields information on the
frequency of adducts. To interpret the blots, one must introduce a
sufficient level of damage to evaluate the P(0). Under our
experimental conditions, 0.3 µM 1-NOP and 1.2 µM N-AcO-AAF resulted in this level of adduct formation/20-kb strand
of the HPRT gene (Table 2). This level of damage reduced
the colony-forming ability (i.e. the survival) of treated
cells to 8% in the case of 1-NOP and 51% in the case of N-AcO-AAF.
These results are in excellent agreement with previous results from
this laboratory correlating survival of colony-forming ability with the
frequency of such adducts induced in the overall genome of excision
repair-proficient fibroblasts as determined using labeled
carcinogen(28, 29) . If the level of adduct formation
in the 5`-half of the HPRT gene is similar to that in the
overall genome, the data indicate that under the conditions used in the
present study, UvrABC recognized and incised the majority of the
adducts induced by either agent.-BP. In contrast, we found
that excision of dG-C8-AF proceeded very slowly, much slower that
dG-N-BP, and was not strand-specific. Yet our data on
interference with transcription (Fig. 5), showed that only 1.6
dG-N-BP were required to reduce transcription to 37% of
that directed by the unmodified template while 6.8-7.0 dG-C8-AP
or dG-C8-AF were required to do so. These data are in good agreement
with the degree of interference with elongation by T7 RNApolymerase
presented by site specifically placed dG-N-BP(30) ,
or dG-C8-AF(31) . Caution must be exercised in extrapolating
from in vitro transcription assays to the behavior of human
RNA polymerases in vivo. However, Sorcher and Cordeiro-Stone (32) showed that the absence of excision repair, 2.1
dG-N-BP reduced transcription by human RNA polymerases of a
transfected reporter gene to 37% of control. This is in agreement with
the value of 1.6 found in the in vitro assay reported in this
study for the same adduct. Xenopus RNA polymerase III has been
reported to be blocked to a greater extent by site-specifically placed
dG-C8-AF than predicted from in vitro assays(33) .
However, if human RNA polymerase II were also blocked by such lesions
to a greater extent than predicted, our conclusions would still hold,
namely, that the rate at which cells carry out preferential,
strand-specific repair is not determined solely by the degree to which
the adducts being excised block transcription. nucleotides reduces the surviving fraction of a
population of treated cells to 37% (29) ; dG-C8-AF is much less
cytotoxic, i.e. 50 such adducts/10 nucleotides are
needed to reduce survival to 37% in these cells(28) . What is
more, at a level of 25 adducts/10 nucleotides, dG-C8-AP
yields 4 times as many HPRT mutants as
dG-C8-AF(28, 37) . position of guanine, a site of base
pairing, but the reorientation of the adducted guanine causes the
(+)-anti-BPDE adduct to lie in the minor groove, with the
pyrenyl ring oriented toward the 5`-end of the modified strand where it
causes little helical distortion(12, 38) . BPDE
adducts (39) are significantly more cytotoxic and more
mutagenic than dG-C8-AP (29) or dG-C8-AF(37) . Like
dG-C8-AP, BPDE-induced adducts are repaired from the human HPRT gene in a strand-specific manner(3) , although at a rate
2-fold slower than that of dG-C8-AP. (The half-life of 1-NOP adducts in
the transcribed strand was 4 h; in the nontranscribed strand was 8 h;
and in the overall genome was 10 h, compared with 7.5, 15, and 24 h,
respectively, for BPDE adducts.)-guanine adduct formed by BPDE. However, this
model does not explain why an adduct such as dG-C8-AP, which lies in
the major groove and does not interfere with base pairing, is repaired
so efficiently, nor why the structurally related dG-C8-AF is repaired
so poorly. Our data indicate that the rate and mode (i.e. global versus transcription-coupled mechanisms) of
excision repair are determined by a complex interplay of adduct
structure, its accessibility to repair enzymes, its ability to arrest
transcription, and local DNA conformation.
)-BP, N-deoxyguanosinyl-benzo(a)pyrene;
dG-C8-AP, N-(deoxyguanosin-8-yl)-1-aminopyrene; dG-C8-AF, N-(deoxyguanosin-8-yl)-2-aminofluorene; kb, kilobases ;
N-OH-AF, N-hydroxy-2-aminofluorene.
We thank Dr. P. van de Putte for generously supplying
UvrABC and Dr. Fred Beland for generously supplying tritiated BPDE,
1-nitrosopyrene, and N-hydroxy-2-aminofluorene as well as
discussions on optimal reaction conditions. We also thank our
colleague, Beatrice S. Tung, for helpful discussions about this
research.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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