[CANCER RESEARCH54, 175-t81, January I, 1994]
Ongoing Activity of RNA Polymerase II Confers Preferential Repair
of Nitrogen Mustard-induced N-Alkylpurines in the Hamster
Dihydrofolate Reductase Gene I
Karsten Wassermann 2 and Jesper Damgaard
Department of Toxicology and Biology, National Institute of Occupational Health, Denmark, Lersr Parkalld 105, DK-2100 Copenhagen, Denmark
ABSTRACT
Recently, it has been demonstrated that nitrogen mustard-induced Nalkylpurines are excised rapidly from actively transcribing genes, while
they persist longer in noncoding regions and in the genome overall. It was
suggested that transcriptional activity is implicated as a regulatory element in the efficient removal of lesions. By treating cells or not with the
transcription inhibitor a-amanitin, we have explored whether ongoing
activity of RNA polymerase II was coordinately related to proficient repair
of nitrogen mustard-induced alkylation products in the actively transcribed dihydrofolate reductase gene in the Chinese hamster ovary B l l
cells. Nuclear run-off transcription analysis verified that ~-amanitin completely and selectively inhibited transcription by RNA polymerase II. At
the drug exposure examined, nitrogen mustard induced DNA damage
capable of a complete transcription termination in the RNA polymerase
II-transcribed dihydrofolate reductase gene and reduced 28S rDNA transcription by a factor of 7.9. The transcription activity did partially recover
following reincubation in drug-free medium; this recovery was about 34
and 76% of ribosomal 28S gene transcripts and dihydrofolate reductase
gene transcripts, respectively, after 6 h of repair incubation, a-Amanitin
significantly inhibited the removal of nitrogen mustard-induced N-alkylpurines in the 5'-half of the essential, constitutively active dihydrofolate
reductase gene, while no effect of a-amanitin was observed on the lesion
removal from a noncoding region 3'-flanking to the gene and from the
genome overall. In the actively transcribed gene region, about 77% of
N-alkylpurines were removed 21 h following drug exposure of cells not
treated with a-amanitin and about 47% in 21 h in a-amanitin treated
cells. The global semiconservative replication seemed unaffected by the
a-amanitin treatment. From these results we suggest that gene-specific
repair of nitrogen mustard-induced N-alkylpurines is dependent on ongoing activity of the transcribing RNA polymerase II. The findings are
discussed in terms of the current ideas about the mechanism of preferential DNA repair.
INTRODUCTION
Recent development of various techniques to examine the formation and the repair of various DNA lesions in individual genes has
advanced our knowledge of the DNA repair processes in a wide array
of organisms (see Ref. 1 for review). Especially in the case of UV
light-induced cyclobutane pyrimidine dimers, ample evidence has
been presented that repair of this lesion occurs preferentially in transcriptionally active genes (2-5). Also, the efficiency of DNA repair
may be affected by the nature of the lesions, its location within the
genome, the transcriptional activity, and the conformation of the chromatin structure of the damaged DNA regions.
DNA base damage is known to undergo a complex excision repair
mechanism involving several specific enzymes (6). Considerable differences in the mode of repair exist among different monofunctional
alkylating agents and even among agents which cause similar types of
DNA lesions. DNA base methylation introduced with dimethyl sulfate
Received 7/28/93; accepted 11/1/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by the Danish Cancer Society (Grant 92-030).
2 To whom correspondence should be addressed.
seem to be randomly removed from the genome independently of its
location within the genome (7, 8), while a correlation between transcriptional activity and availability to lesion removal of methylnitrosourea-induced DNA methylation has been demonstrated in the same
hamster cells (9), a rat insulinoma cell line (10), and cultured human
cells (11). Although alkylation by SNl-alkylating agents (e.g., methylnitrosourea) has greater sequence selectivity than Sy2-alkylating
agents (e.g., dimethyl sulfate) in vitro (12), it is not clear how this
would result in differences in the mode of repair. It is likely that the
observed differences in repair of methylpurines may depend on the
degree of chromatin distortion invoked by these monofunctional alkylating agents and thereby the recognition enzymes involved. Alternatively, it may reflect the degree of transcription-terminating lesions
induced by the DNA adducts.
A particularly intriguing problem is to establish the relationship, if
any, between DNA repair and transcription. HN23 is a highly electrophilic anticancer agent which alkylates DNA bases mainly at N 7guanine and to a much lesser extent at other positions (13-15). HN2
bears two alkylating groups per molecule which together allow the
formation of cross-links between paired DNA strands, between guanines in the same strand, or between DNA and protein (16-20). The
biological effect of HN2 may depend on preferential reaction at certain genomic locations. Recently, we described a general approach to
analyze gene-specific damage and repair of N-alkylpurines; the methodology is especially suitable for measuring NT-guanine alkylation
upon exposure in vivo and measures the production of both DNA
monoadducts and cross-links (8). We have shown that HN2-mediated
N-alkylpurines are excised rapidly from actively transcribing genes
but persist longer in an inactive gene region and a noncoding region
of the hamster genome (8, 21). Also, the much less frequent HN2mediated DNA interstrand cross-links have recently been reported to
be preferentially repaired (22, 23). Finally, by using the same general
technique for the measurements of N-alkylpurines (8), but probing the
membranes with single-stranded RNA probes (riboprobes), we observed a slight bias toward repair in the transcribed strand after exposure to HN2. Thus, the phenomenon of preferential repair of HN2induced N-alkylpurines resemble that seen for the removal of UV
light-induced cyclobutane pyrimidine dimers, although some quantitative differences may occur (1).
The biological advantage of preferential repair is evident, but the
mechanism by which it occurs is still largely unknown. The observation that repair of HN2-induced alkylation products occurs nonrandomly in the genome suggests that the transcription complex could be
actively involved in the recognition and removal of HN2-induced
N-alkylpurines. The lesions generated by HN2 capable of blocking
transcription at the NT-guanine alkylation sites in DNA (24) may be an
important signal: stalled RNA polymerase could direct repair enzymes
to the transcription-terminating lesion. To determine whether ongoing
transcription is required for the preferential repair of HN2-induced
DNA lesions, we have used o~-amanitin to inhibit transcription by
RNA polymerase II in HN2-treated CHO cells, a-Amanitin, the bi3 The abbreviations used are: HN2, nitrogen mustard, bis(2-chloroethyl)methylamine;
CHO, Chinese hamster ovary; DHFR, dihydrofolate reductase.
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TRANSCRIPTION-DEPENDENT GENE REPAIR OF HN2 ALKYLATION
cyclic octapeptide from the poisonous mushroom, Amanita phalloides, binds to R N A polymerase II, thereby specifically blocking the
elongation of m R N A transcripts without affecting the binding of the
polymerase to the D N A template (25, 26). We examined the rate and
extent of removal of HN2-induced N-alkylpurines from the actively
R N A polymerase II transcribed D H F R gene, a noncoding region 3'flanking to the D H F R gene, and from total cellular D N A in cells
treated or not with a-amanitin. The effectiveness of transcription
inhibition by a-amanitin and H N 2 was confirmed by nuclear run-off
transcription analysis.
i
pMB5
cs14DO
5.8S
18S~. 28S
rDNA
5' -
-
pAbb
MATERIALS
AND
METHODS
Isotopes, Drugs, Enzymes, and Hybridization Probes. [methyl-3H]-Thy midine (85 Ci/mmol), [c~-32p]dCTP (3000 Ci/mmol), [5,6-3H]-uridine (49 Ci/
mmol), and [c~-32P]UTP(800 Ci/mmol) were purchased from Amersham Denmark. a-Amanitin was purchased from Boehringer Mannheim (Ercopharm,
Denmark). HN2 was obtained through the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, and was kept
as a 0.1 M stock solution in 0.1 M HC1 at -20~ Restriction endonucleases
(KpnI and HindIII) were purchased from New England Biolabs, Inc. All probes
for the DHFR gene (pMB5) and the downstream, noncoding region (cs-14DO)
were kindly provided by V. A. Bohr (National Institute on Aging, National
Institutes of Health, Baltimore, MD) and have been described (27). The pMB5
probe was used to detect a 14-kilobase KpnI fragment of the 5'-half CHO
DHFR, and a subclone of cs-14 (cs-14DO) was used to detect the downstream,
noncoding 14-kilobase KpnI fragment of the DHFR gene (Fig. 1). The cs14DO probe was generated by selecting for fragments which contained a
minimum of repetitive sequences.4 The plasmid pAbb was kindly provided by
P. C. Hanawalt (Stanford University, Stanford, CA). The plasmid contains a
1.4-kilobase insert from human 28S ribosomal DNA sequence (28) homologous to the hamster sequence containing the 5.8S and 28S rDNA sequences in
CHO cells (Fig. 1).
Cell Culture. CHO-Bll cells (29) carrying the amplified DHFR gene were
grown in Ham's F-12 medium without glutamine, glycine, hypoxanthine, and
thymidine (Gibco Laboratories) supplemented with penicillin and streptomycin, 500 nM methotrexate, and 10% dialyzed fetal calf serum (Gibco) in
humidified 5% CO2, 95% air at 37~ Cell cultures for all experiments in this
study were in exponential growth phase at the time of DNA damage.
RNA Synthesis. Cells were incubated with 10 p.g/ml (x-amanitin for 0, 3,
4, 5, 6, and 10 h at 37~ Aliquots of cells were incubated in triplicate with
[5,6-3H]uridine (0.05 p.Ci/ml) during the last hour of treatment with a-amanitin. Incorporation of [3H]uridine into RNA was stopped by aspirating the
radioactive medium followed by addition of ice-cold phosphate-buffered saline. After trypsinization cells were resuspended in ice-cold 10% trichloracetic
acid and 2% (w/v) sodium PPi in distilled water. The resulting acid-insoluble
material was collected onto MiUipore GS 0.22-p.m filter discs. The filters were
washed once with 5 ml of the trichloracetic acid/sodium PPi solution and twice
with 5 ml of 95% ethanol and were then air dried. Filters were placed in
scintillation vials and 1.5 ml of 10 mM Tris, pH 9-1 mM EDTA was added.
Capped vials were incubated at 65~ for 1 h, 10 ml of UniScint BD (National
Diagnostics) was added, and the radioactivity was determined using a Beckman LS 1801 liquid scintillation system.
Nuclear Run-off Transcription. The procedure for transcription of nuclei,
subsequent RNA isolation, blotting, and hybridization were performed as previously described (30, 31). Briefly, nuclei were prepared from CHO-Bll cells:
the control treatment consisted of nuclei treated with 10 /xg/m| ot-amanitin,
with 150 /XMHN2, or with both 10 txg/ml c~-amanitin and 150/XM HN2 as in
the gene repair analysis (see below). Nuclei were isolated by harvesting cells
in ice-cold phosphate-buffered saline with a rubber policeman from Petri
dishes placed on ice, pelleting the cells, vortexing the cells in Nonidet P-40
lysis buffer (10 mM Tris-C1, pH 7.4-10 mM NaC1-3 mM MgCIz-0.5% Nonidet
P-40), repelleting at 500 • g for 5 rain at 4~ vortexing again in Nonidet P-40
buffer, and pelleting once again. The nuclei were resuspended in 200 bL1
glycerol storage buffer (50 mM Tris-C1, pH 8.3-40% glycerol-5 mM MgC12-0.1
mM EDTA) and stored in liquid nitrogen. Nuclear run-off transcription reac-
4 D. Okumoto, unpublished data.
I
I
I
I
I
I
I
-10
0
10
20
30
40
50
kb
Fig. 1. Genomic maps of the hamster DHFR and ribosomalgenes showing the regions
analyzed. The DHFR (top) and ribosomal(bottom)genes are shown with KpnI restriction
sites indicated by vertical lines and the coding region by the rectangular horizontalbox
with the positions of the exons. The DHFR-codingregion analyzedis a 14-kilobaseKpnI
fragment (pMB5) and the noncodingregion analyzed is also a 14-kilobaseKpnI fragment
(csl4DO). The ribosomal gene region analyzed is a 9-kilobase KpnI fragment (pAbb)
containing the 28S and 5.8S gene regions.
tions were carded out with 200 p.1 thawed nuclei (107 nuclei per sample), 200
p.l of 2x reaction buffer (10 mM Tris-Cl, pH 8.0-5 mM MgC12-0.3 M KC1), 2.5
mM dithiothreitol, 100 p.Ci [ot-3zp]uTP, and 0.5 mM ATP, CTP, and GTP.
Transcripts were isolated as described. The entire transcription products ([32p]_
nRNA) were hybridized at 65~ for 36 h to plasmid DNA" pMB5 to detect
DHFR transcripts and pAbb to detect ribosomal gene transcripts. Linearized,
denatured plasmid (4 p.g/slot) was immobilized on a nylon membrane (Amersham Hybond-N § with a slot blot apparatus (Hoefer Scientific Instruments).
The membranes were prehybridized (QuikHyb hybridization solution; Stratagene) as for a Southern blot. After hybridization with the transcription products, membranes were washed twice with 2• standard saline citrate (1 • is 150
mM NaCI-15 mM trisodium citrate, pH 7.0) for 1 h each at 65~ incubated with
0.01 mg/ml RNase for 30 min at 37~ washed again once in 2• standard
saline citrate for 1 h at 37~ and then autoradiographed.
Gene Repair Analysis. Cellular DNA was prelabeled by allowing cells to
grow for 64 h at 37~ in [3H]thymidine (0.3 /xCi/ml) and 10 ~M "cold"
thymidine. Following this procedure, the cells were subcultured and incubated
in label-free medium for 24 h. The steps involved in the measurement of DNA
damage and repair in specific genomic regions have been described in detail
(8). Briefly, the prelabeled cells were treated with 10 p,g/ml ct-amanitin for 6
h in fresh medium prior to an exposure to 150/XM HN2 for 30 min at 37~ in
fresh medium supplemented with 1% fetal calf serum. At the end of drug
exposure, cells were washed with phosphate-buffered saline and either lysed
(16 h at 37~ pH 9.0) immediately or after repair incubation. For repair, the
a-amanitin-containing medium was returned to the culture, which contained
bromodeoxyuridine and fluorodeoxyuridine, to density label DNA replicated
during the repair incubation. Control cultures received no a-amanitin. High
molecular weight DNA was isolated by high-concentration salt extraction (32)
and then treated with the appropriate restriction endonuclease. The parental
DNA was then separated from that which had been replicated during repair
incubation by neutral CsCI density gradient centrifugation (Fig. 2).
Samples containing 5/xg of purified parental DNA were depurinated at all
heat-labile alkylated bases and then hydrolyzed in alkali to cleave the DNA at
apurinic sites. Plasmid pBR322, linearized by digestion with HindlII, was
added to the individual samples after the alkaline hydrolysis. This served as an
internal standard to control for the relative quantitation in individual samples.
Duplicate DNA samples, each containing 2.5 p.g of DNA, were subjected to
electrophoresis in 0.5% alkaline agarose gels, Southern transferred onto nylon
membranes (Amersham Hybond-N+), prehybridized, and hybridized to specific 3Zp-labeled probes of interest. Autoradiography and densitometry (Shimadzu CS-9001PC dual-wavelength scanner) were performed to determine the
extent of repair (8).
Overall Genome Repair. Repair of alkylation damage in total cellular
DNA was determined by a gel electrophoresis method using densitometer scan
of photographic negatives of ethidium bromide-stained agarose gels. Detailed
methodology can be found elsewhere (8, 33). Samples of total parental cellular
DNA were prepared as described above in order to convert drug-induced
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TRANSCRIPTION-DEPENDENT GENE REPAIR OF HN2 ALKYLATION
A
b 3
X
O
0 2 4 6 8 10 12 14 16 18 20
Fraction number
0
2 4
6 8 10 12 14 16 18 20
Fraction number
Fig. 2. Neutral CsCI gradient prof'des of genomic DNA. Cellular DNA from CHO-B11
cells were prelabeled with [3H]thymidine and incubated with 10/zg/ml ot-amanitin or not
6 h before and after 150/xM HN2 exposure for 30 rain. For repair, cells were incubated
in medium containing 10 /.~Mbromodeoxyuridine and 1 /~M fluorodeoxyuridine. Total
cellular KpnI-restricted DNA was sedimented in neutral CsC1 gradients and fractionated.
Graphs represent the total radioactivity in each fraction containing parental and densitylabeled replicated DNA. A, DNA from cells 0 h after HN2 exposure; B, DNA from cells
6 h after HN2 exposure. O, control; A, 150 p,M HN2; @, 10 p,g/ml ct-amanitin; A, 10
/xg/ml ot-amanitin and 150 p,M HN2.
alkylation to single-strand breaks. Alkaline samples were then subjected to
electrophoresis in 0.5% alkaline agarose gels, which was the same gel used for
gene repair analysis after processing. The ethidium bromide-stained D N A from
destained agarose gels was photographed on a UV transilluminator with Polaroid type 55 film. The distribution of the D N A along each lane in the agarose
gels was then determined by scanning the photographic negative and was
analyzed using a Shimadzu CS-9001PC dual-wavelength scanner. The average
molecular weight and repair percentage were calculated by the use of HindlIIdigested A-phage DNA as a standard. The migration of this standard was
scanned as was the D N A from experimental samples.
very high transcription levels as compared to the RNA polymerase
II-transcribed DHFR gene (Fig. 4A). Exposure to ct-amanitin confirmed that RNA polymerase II is highly sensitive, whereas RNA
polymerase I is resistant to this inhibitor (Fig. 4B). Yet, complete
inhibition of DHFR transcription by a-amanitin obtained after 6 h of
exposure to 10 p,g/ml partially reversed upon removal and reincubation for 6 h in ct-amanitin-free medium (Fig. 4C); this recovery was
calculated to be 20%. Treatment of cells with 150/~M HN2 for 30 min
caused a severe block of DHFR transcription and significantly reduced ribosomal gene transcription by a factor of 7.9 (Fig. 4D); this
was revealed by densitometry of an autoradiogram. It appeared that 6
h of reincubation of HN2-treated cells in a-amanitin- and drug-free
medium caused a partial recovery of both ribosomal gene and DHFR
gene transcription (Fig. 4E); this recovery was calculated to 34.4 and
75.9% for the ribosomal gene and DHFR gene, respectively. The
regain of RNA polymerase II-mediated transcription was sensitive to
a-amanitin (Fig. 4F).
Effect of a-Amanitin on Removal of N.Alkylpurines in the
DHFR Gene and in Its 3'-Flanking Noncoding Region. HN2-induced N-alkylpurines are removed preferentially from the actively
transcribed DHFR gene in various CHO cells (8, 21). To determine
whether this preferential repair is dependent on ongoing transcription,
we compared the rate of removal of HN2-induced N-alkylpurines
from specific genomic regions in the CHO-Bll cells by use of the
established procedure described by Wassermann et aL (8). After treatment of cells with 150/~M HN2 alone or of cells preincubated with 10
p,g/ml a-amanitin, repair incubation for 0, 6, and 21 h, isolation, and
purification of parental DNA as described, the repair kinetics were
determined in the 5'-half of the DHFR gene and in the 3'-flanking
noncoding region of the DHFR gene. In order to secure that heating
of untreated purified DNA did not cause nonspecific depurination,
100
RESULTS
Effect of a-Amanitin and Nitrogen Mustard on Ongoing Tran.
scription. Cell cultures were pretreated with ct-amanitin to induce
complete blockage of transcription by the time of drug treatment and
subsequent repair incubation. To determine the optimal exposure time
of this pretreatment we began measuring the time-dependent effect of
a-amanitin on total RNA synthesis. Within 6 h, 10 ~g/ml c~-amanitin
reduced precursor incorporation into RNA to about 50% of that observed in untreated control cells (Fig. 3). Because the level of 3H
activity of acid-insoluble material did not further decrease upon longer
exposure to ot-amanitin, we chose 6 h as the time of pretreatment to
ensure maximal inhibition of RNA synthesis. The residual activity of
RNA synthesis reflects rRNA transcription since RNA polymerase I is
resistant to ct-amanitin (34).
To examine whether ot-amanitin selectively induced stalled RNA
polymerase II-mediated transcription complexes, we performed a
nuclear run-off transcription assay. In this in vitro assay, new RNA
transcripts are not initiated, but transcripts which are already initiated
are faithfully elongated, giving a reasonably accurate measure of the
level of transcription at any time chosen and from any gene of interest.
Since a-amanitin induced a 50% reduction of total RNA synthesis, we
could not assume that the overall level of RNA synthesis was constant
and independent of the function of cell state (31). Therefore, as we
examined the effect of both a-amanitin and HN2 on transcription, we
hybridized the entire run-off product from the same number of nuclei
in all samples.
The results from the nuclear run-off analysis are shown in Fig. 4.
The several hundred copies of the ribosomal 28S genes resulted in
.1..,
tO
80
"6
v
t-
.o
.,...
60
o
o
o
_.= 40
r
t-o
.m
fO
20
0
k
I
I
I
I
I
I
I
1
)
I
0 1 2 3 4 5 6 7 8 9 1 0
Time of Incubation (hours)
Fig. 3. Effect of ot-amanitin on total RNA synthesis in Chinese hamster ovary cells.
CHO-Bll cells exposed to 10/~g/ml a-amanitin for various times at 37~ and untreated
control cells were labeled for a 1-h period with [3H]uridine. Acid-insoluble material was
collected, and radioactivity was determined by liquid scintillation counting. Results are
expressed as the percentage of [5,6-3H]uridine incorporated by ot-amanitin-exposed cells
in comparison to that by control cells. Data are the means --- SE from five individual
experiments.
177
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TRANSCRIPTION-DEPENDENT GENE REPAIR OF HN2 ALKYLATION
duplicate samples of nonheated DNA from untreated cells were either
loaded directly into the alkaline agarose gel or subjected to neutral
depurination (heating at 70~ for 30 rain at pH 7.0), followed by
A
alkaline hydrolysis (0.1 N NaOH for 30 rain at 37~ prior to electrophoresis (Fig. 5). A decrease in the amount of full-length restriction
fragment in the heated control compared to the nonheated sample
represents nonspecific depurination. From repeated experiments, this
nonspecific cleavage was estimated to be <10%.
Quantitation of the autoradiograms revealed that a-amanitin did not
C
interfere significantly with the initial lesion frequency in either the
DHFR gene or the noncoding region (0.05 < P < 0.1, t test) (Fig. 5,
Table 1). Although small differences was seen in the formation of
D
N-alkylpurines, one should interpret these differences with caution
since there was considerable variation among experiments; this was
mainly due to variation in the precise measurements of the low band
intensities. For the DHFR gene, efficient repair of N-alkylpurines
appeared by 6 h in cells not treated with a-amanitin (Fig. 5, Table 1).
The removal of HN2-induced lesions in the DHFR region from a-amanitin-treated cells, however, was significantly less (Fig. 5, Table 1).
Thus, in the 14-kilobase 5'-half DHFR region about 57 and 77% of
N-alkylpurines were repaired in 6 and 21 h, respectively, in cells not
treated with a-amanitin and 23 and 47% in 6 and 21 h, respectively,
in ot-amanitin-treated cells (Fig. 5, Table 1). The removal of alkali0,8
labile lesions from the nontranscribing 3'-flanking region of the
DHFR gene confirmed that HN2-induced N-alkylpurines are preferentially repaired because they were considerably less efficiently re"~. 0,6
moved than in the DHFR gene (Fig. 5, Table 1). The repair of the
noncoding region was not affected by treatment of the cells with
0,4
ot-amanitin (Fig. 5, Table 1).
Effect of a-Amanitin on the Removal of N-Alkylpurines from
Total Cellular DNA. The overall genome repair was determined in
order to define whether a-amanitin grossly affected the repair system
active in the removal of HN2-induced N-alkylpurines in addition to its
0
demonstrated inhibition of RNA polymerase II. By measuring the
A
B
C
D
E
F
overall genome repair from the agarose gels, which also was used for
Fig. 4. Effect of ot-amanitin and HN2 on ongoing transcription. CHO-Bll cells were
the gene analysis, we could compare overall genome- and geneexposed to different conditions: A, untreated control; B, 10/~g/ml o~-amanitin for 12 h at
37~ C, 10 p~g/ml a-amanitin for 6 h at 37~ followed by 6 h at 37~ in a-amanitin-free
specific repair on the same biological sample. Electrophoresis of
medium; D, 150 ~ra HN2 for 30 min at 37~ E, 150/xM HN2 for 30 min at 37~ followed
DNAs on agarose gels disperses DNA according to molecular length.
by reincubation for 6 h in drug-free at 37~ F, 10 ~g/ml a-amanitin for 12 h and 150/~M
A typical photographic negative of an alkaline agarose gel used for
HN2 for 30 rain at 37~ where nuclei were isolated 6 h after exposure to HN2. Top,
32p-labeled nuclear run-off transcription products ([32p]nRNA) were hybridized to nylonoverall DNA repair by the alkaline gel electrophoresis method is
bound plasmid DNA: pAbb to detect ribosomal 28S gene transcripts and pMB5 to detect
shown in Fig. 6. Lane L contained the molecular length marker
5'-half of DHFR transcripts. Bottom, data derived from densitometry analysis of autoradiograms of nuclear run-off transcription products. I , ribosomal 28S gene; [~, 5'-half of
(HindIII-digested)~-phage DNA) as a standard used for the calculathe DHFR gene.
tions of the average molecular weight and repair percentages of the
pAbb pMB5
g
5
_~.. ~ l
A
f ~ ' ~ " [ l '
5"- half DHFR
B
C
Ii
D
ii
E
It'
tmIPm
"
F
ii
G
ii
H
I|
I
II
J
|1
I
qwi~
"
3" fla ki g
Fig. 5. Effect of a-amanitin on repair of HN2-induced N-atkylpurines in the CHO DHFR gene. DNA from CHO-BI 1 cells that had been treated with 10/xg/ml a-amanitin or not
prior to and after exposure to 150/XM HN2 for 30 min at 37~ were digested with Kpnl. Autoradiography of Southern blots detected hybridization of 32p-labeled genomic probes made
from pMB5 to the 14-kilobase KpnI fragment of the 5'-half of the DHFR gene and cs-14DO to the 14-kilobase KpnI fragment of the 3'-flanking noncoding region. Lanes A and B,
duplicate samples of DNA from nonheated (nondepurinated) control and heated (depurinated) control, respectively. Lanes C-J, duplicate samples of DNA from cells treated with: 150
/.zM HN2 for 30 rain at 37~ and repair incubated for 0 h (C), 6 h (D), and 21 h (H); 10 ~g/ml a-amanitin 6 h prior to a treatment with 150 /xM HN2 for 30 min at 37~ and during
repair incubation for 0 h (E), 6 h (F), and 21 h (1); 10/xg/ml a-amanitin for 12 h (G) and 27 h (J) as a 6- and 21-h a-amanitin repair control, respectively.
178
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TRANSCRIPTION-DEPENDENT GENE REPAIR OF HN2 ALKYLATION
Table 1 Effect of a-amanitin on repair of HN2-induced alkali-labile lesions in different regions of the CHO genome
Data were derived from densitometry analysis of autoradiograms of Southern blots and are presented as mean values • SE from three individual experiments.
Probe region
5'-half DHFR
3' flanking
Time
(h)
0
6
21
0
6
21
HN2 (150 txM)
(lesion/10 kilobases)
0.83
0.35
0.19
0.66
0.48
0.28
% HN2 lesions removed
[(-) a-amanitin]
___0.10
• 0.07
• 0.03
--- 0.06
--- 0.04
• 0.04
0
57.8
77.1
0
27.3
57.6
HN2 (150/.LM) +
a-amanitin (10/zg/ml)
(lesions/10 kilobases)
0.63
0.48
0.33
0.90
0.55
0.34
• 0.07
• 0.02
• 0.10
--- 0.15
• 0.11
• 0.11
% HN2 lesions removed
[(+) ol-amanitin]
0
23.8 a
47.6 a
0
38.9
62.2
a p = 0.05, Wilcoxon test.
individual DNA samples. The mobility of DNA from cells treated with
150/ZM HN2 was increased and was distributed toward the lower part
of the gel indicating alkylation, which had been converted to strand
breaks with a subsequent lower average molecular weight (lanes C
and D). As cells were allowed to repair, their DNA distribution profiles accordingly indicated an increase in the average length of DNA.
This was evident as a slower migration in the electrophoresis (lanes E,
F, H, and I). Within 6 h of repair incubation, 19 and 21% of the
alkylation adducts had been removed from cells treated with HN2
alone or with HN2 and a-amanitin, respectively. After 21 h, about
51% had been removed from cells treated with HN2 but not with
a-amanitin and 41% in cells treated with both HN2 and a-amanitin.
Therefore, global repair processes active in the removal of HN2induced N-alkylpurines were not significantly affected by ot-amanitin.
somal probe sequence was significantly reduced immediately following exposure to HN2. Since ribosomal genes of mammalian cells are
known to be significantly enriched in G + C content with several large
segments containing >80% G + C (36, 37), the transcription of these
genes impeded was as expected by HN2 alkylation. These observations further add to other reports demonstrating that transcribing polymerases stall at bulky lesions on the DNA template (38--42).
The ot-amanitin experiments showed obliteration of preferential
repair of HN2-induced N-alkylpurines in the actively transcribed
DHFR gene. c~-Amanitin had no effect on repair in the noncoding
region. The combined results suggest that the transcriptional complex
can direct repair enzymes to the DNA lesions possibly because these
act as a termination site of RNA polymerase or at least stall its
progress. Alternatively, the observed differences in repair could be
related to a unique or enhanced alteration in the DNA secondary
DISCUSSION
structure, which targets the repair enzyme to the site of lesion. Moreover, HN2 and other bulky adducts, which cause termination of tranRecent evidence suggests that N-alkylpurines produced by HN2 are scription, may provoke a buildup of truncated transcripts or stalled
processed in a nonrandom fashion in the mammalian genome and that
transcription complexes which further could distort the DNA and act
these alkylation lesions are more efficiently removed from actively
as a signal for the repair enzymes. Either scenario would explain the
transcribing genes than from noncoding regions and from the genome
preferential removal of HN2-induced N-alkylpurines from the actively
overall (8, 21-23). It was suggested that transcriptional activity is
transcribed DHFR locus. The recent experiments by May et al. (43)
implicated as a regulatory element in the availability of efficient lesion
showed a slight strand bias toward repair of HN2-induced N-alkylpuremoval.
rines in the transcribed strand of the hamster DHFR gene. Although
By treating cells or not with the transcription inhibitor ot-amanitin
these studies attempted to examine a role for transcription in preferwe have explored whether RNA polymerase II activity was coordiential repair, they did not reveal any direct role of participating trannately related to proficient repair of HN2 alkylation products in an
scription factors. While the observed difference of the repair rate
actively transcribed gene. The findings presented in this report show
between the template and complementary strand, however, was less
that a-amanitin markedly inhibited the removal of HN2-induced Nthan the a-amanitin-sensitive fraction in this study, both studies point
alkylpurines in the 5'-half of the essential, constitutively active DHFR
gene, while no effect of a-amanitin was observed on the lesion re- to a role for transcription in gene-specific repair.
Although provocative, the use of the elongation-specific inhibitor
moval from a noncoding region 3'-flanking to the DHFR gene and the
oL-amanitin
may also leave open an alternative model: preferential
genome overall. Nuclear run-off transcription reactions verified that
repair
requires
the continuous expression (i.e., transcription translaa-amanitin completely and selectively inhibited transcription by RNA
tion)
of
a
very
labile
or unstable "coupling" factor. Such a drastically
polymerase II.
different
model
may
be supported by the fact that a-amanitin is not
The nuclear run-off experiments revealed a profound HN2-mediated inhibition of transcription of both the DHFR gene and rDNA. The selective for the inhibition of RNA polymerase II transcribing the
hamster DHFR gene has several G-rich GC boxes proximal to the DHFR gene but may inhibit the elongation step mediated by any RNA
major DHFR transcription start site and several GC boxes of the polymerase II. Thus, the inhibition of gene-specific repair may alteropposite orientation (C-rich) in a distal region upstream (35). The first natively, or additionally, result from inhibition of transcription of this
coupling factor rather than inhibition of DHFR transcription per se.
exons lie within a region marked by an exceptionally high G + C
content and lack of DNA methylation, which correlates with transcrip- Within this context, it is interesting that an intriguing transcriptiontional activity (35). At 150 /.~M HN2, we detected less than one coupling factor that conferred gene- and strand-specific repair of UV,
alkali-labile lesion per 10 kilobases in the DHFR probe region. Since psoralen, and cisplatin-induced damage recently was partially purified
(44). This important finding further supports the notion that a protein
regions of the genome rich in contiguous guanines are considered
preferential target sites for HN2, one would expect the 5' portion of couples transcription and repair; yet the actual candidate(s), remains
the gene to contain a large fraction of this damage at or near the to be identified.
The treatment of cells with a-amanitin had no discernible effect on
promotor sequence. Although the exact sequence of the sites of lesions
are unknown, other studies have shown that HN2 produces transcrip- the removal of N-alkylpurines from the genome overall. This suggests,
tion-terminating lesions at or near guanine pairs in vitro (24) and that that genes which are actively transcribed by RNA polymerase II must
the DNA sequence selectivity of N7-guanine alkylation by HN2 is represent a small component of the total cellular genome. Furtherpreserved in intact cells (15). Also, ongoing transcription of the ribo- more, the neutral CsC1 gradient profiles revealed that the global semi179
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer
Research.
TRANSCRIPTION-DEPENDENT
A
L
T
A
,
B C D
,,
11
lw
E F G H
~,
, f~-'l~
I
I I~""I
i
GENE REPAIR OF HN2 ALKYLATION
strand is mediated by e n z y m e s associated with the transcription machinery. While w e cannot completely rule out an indirect effect of the
ot-amanitin-mediated transcription b l o c k on c h r o m a t i n structure, the
simplest interpretation of our results is also a transcription-mediated
direction of repair toward lesions in the D N A . Yet, a c o m b i n a t i o n of
cellular factors that help repair c o m p l e x e s to find and correct the
lesions m a y exist. Nonetheless, as HN2-treated cells w e r e allowed to
incubate in drug- and a-amanitin-free m e d i u m , the R N A p o l y m e r a s e
II r e s u m e d to a remarkable level (75.9% of control), w h i c h allowed
the active gene region to be efficiently repaired.
E x a m i n i n g transcription activity by nuclear run-off analysis provides information c o n c e r n i n g the o n g o i n g transcription and may, in
turn, afford valuable information regarding the processing of D N A
lesions introduced into the gene fragment of interest. As such, the
run-off analysis invites speculation that H N 2 - i n d u c e d N-alkylpurines
m a y remain in ribosomal genes, in contrast to in the 5 ' - h a l f o f the
D H F R gene, because only a m i n o r recovery o f transcription products
was observed u p o n repair incubation (Fig. 4). It is important to consider w h e n interpreting data derived f r o m nuclear run-off analysis,
however, that inhibition of transcription run-off d e p e n d s on the distance between the p r o m o t o r and R N A probe as well as the lesion
frequency within this gene segment. Such factors are important for
c o m p a r i s o n o f transcription inhibition of different genes and should be
further explored. Yet, our findings correlate with previous reports
s h o w i n g inefficient r e m o v a l of psoralen p h o t o a d d u c t s (47), pyrimidine dimers, and H N 2 alkylation (48) f r o m r R N A genes.
Recently, other studies have s h o w n that oL-amanitin reduces preferential repair of U V - i n d u c e d p y r i m i d i n e dimers in m a m m a l i a n cells
(45, 49, 50); yet, only in the study by Christians and Hanawalt (45) did
the authors verify the selective effect of tx-amanitin on R N A p o l y m erase II. In the case of H N 2 - i n d u c e d N-alkylpurines w e have s h o w n
that o n g o i n g activity of R N A p o l y m e r a s e II confers preferential repair
in the hamster D H F R gene, but questions still r e m a i n regarding h o w
the transcriptional c o m p l e x directs repair to the lesion.
J
,r
9
23.1 -
9.4-6.64.4-
2.32.0-
0.56 -
B
100
80
.r,_
60
r
E
40
Q.
20
l
0
I
]
l
I
I
I
I
6
I
I
~
I
l
21
ACKNOWLEDGMENTS
Time of Repair Incubation (hours)
Fig. 6. Effect of cz-amanitinon the removal of HN2-induced N-alkylpurines from CHO
cells. DNA from CHO-Bll cells that had been treated with 10/xg/ml c~-amanitinor not
prior to and after exposure to 150 V~MHN2 for 30 min at 37~ were digested with KpnI
and subjected to alkaline agarose gel electrophoresis. Top, photographic negative of an
ethidium-stained agarose gel depicting the distribution of total cellular DNA, Lanes A and
B, DNA in duplicate from nonheated (nondepurinated) control and heated (depurinated)
control, respectively. Lanes C, E, and H, DNA from cells treated with 150/~i HN2 for 30
rain and repair incubated for 0 h (C), 6 h (E), and 21 h (H). Lanes D, F, and I, DNA from
cells treated with 10 t~g/ml ~z-amanitin 6 h prior to a treatment with 150/xm HN2 for 30
rain and repair incubated in the presence of 10/,zg/ml c~-amanitinfor 0 h (D), 6 h (F), and
21 h (I). Lanes F and J, DNA from cells treated with 10/xg/ml ot-amanitin for 12 and 27
h, respectively. Lane L, A-phageHindIIl DNA marker. Bottom, data derived from densitometry analysis of DNA distribution obtained after electrophoresis in alkaline agarose
gels and presented as mean values -+ SE from four individual experiments. &, 150 ~xi
HN2; O, 10 ~g/ml a-amanitin and 150 #,M HN2.
conservative replication processes s e e m e d not to be significantly affected by the ot-amanitin treatment. Similar observations have been
m a d e c o n c e r n i n g the processing of U V - i n d u c e d D N A d a m a g e in the
overall g e n o m e (45). Therefore, w e conclude that the effect o f a - a m anitin was c o n f i n e d to the m o r e efficient repair of the active gene
region.
Our experiments did not address the issue of local topology, " o p e n "
c h r o m a t i n c o n f o r m a t i o n of transcribed D N A , w h i c h m a y be m o r e
accessible than inactive D N A to repair enzymes. While this m o d e l
initially could explain the p h e n o m e n o n of preferential repair of active
genes, it was later challenged by evidence o f preferential repair of
U V - i n d u c e d p y r i m i d i n e dimers in the transcribed strand (4, 5, 43, 44,
46) and o f H N 2 - i n d u c e d N-alkylpurines (43). Rather, strand-specific
repair m a y indicate that an increased rate of repair in the template
We are grateful to Dr. Bj0m A. Nexo for stimulating discussions and for his
comments on the manuscript. We especially thank Drs. P. C Hanawalt for
providing the plasmid pAbb and V. A. Bohr for providing the plasmids pMB5
and cs-14DO.
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Research.
Ongoing Activity of RNA Polymerase II Confers Preferential
Repair of Nitrogen Mustard-induced N-Alkylpurines in the
Hamster Dihydrofolate Reductase Gene
Karsten Wassermann and Jesper Damgaard
Cancer Res 1994;54:175-181.
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