(Received for publication, October 30, 1995)
From the
A prominent model for the mechanism of transcription-coupled DNA repair proposes that an arrested RNA polymerase directs the nucleotide excision repair complex to the transcription-blocking lesion. The specific role for RNA polymerase II in this mechanism can be examined by comparing the extent of polymerase arrest with the extent of transcription-coupled repair for a specific DNA lesion. Previously we reported that a cyclobutane pyrimidine dimer that is repaired preferentially in transcribed genes is a strong block to transcript elongation by RNA pol II (Donahue, B. A., Yin, S., Taylor, J.-S., Reines, D., and Hanawalt, P. C.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8502-8506). Here we report the extent of RNA polymerase II arrest by the C-8 guanine DNA adduct formed by N-2-aminofluorene, a lesion that does not appear to be preferentially repaired. Templates for an in vitro transcription assay were constructed with either an N-2-aminofluorene adduct or the helix-distorting N-2-acetylaminofluorene adduct situated at a specific site downstream from the major late promoter of adenovirus. Consistent with the model for transcription-coupled repair, an aminofluorene adduct located on the transcribed strand was a weak pause site for RNA polymerase II. An acetylaminofluorene adduct located on the transcribed strand was an absolute block to transcriptional elongation. Either adduct located on the nontranscribed strand enhanced polymerase arrest at a nearby sequence-specific pause site.
The cellular processes of replication and transcription can be disrupted by the presence of bulky and helix-distorting lesions in the DNA. Such disruptions may cause cell death or mutagenic processes leading to tumorigenesis. Most if not all organisms utilize the nucleotide excision repair pathway to remove these harmful lesions and restore DNA integrity. An intriguing feature of this pathway is that it can be coupled to transcription. For some lesions the transcribed strands of active genes are repaired at a faster rate than the nontranscribed strands or unexpressed DNA sequences(1) . Transcription-coupled repair provides a mechanism by which the expression of essential genes may be rapidly restored following DNA damage, thereby enhancing cell survival. Indeed, cells from patients with the disorder Cockayne's syndrome lack transcription-coupled repair and exhibit an increased sensitivity to the lethal effects of ultraviolet radiation(2, 3) .
Although the
mechanism of transcription-coupled repair in eukaryotes is not yet
understood, it is clear that the process requires an active RNA
polymerase II (RNA pol II) ()elongation complex. A model has
emerged in which a repair complex is directed to a
transcription-blocking lesion by the arrested RNA pol
II(1, 4) . In an effort to understand the role of RNA
pol II in transcription-coupled repair, we showed that a cyclobutane
pyrimidine dimer, the major DNA lesion formed by short wave UV light,
is a strong block to RNA pol II (5) . The arrested polymerase
is stable and competent to resume elongation, but it prevents
recognition of the CPD by a bacterial repair protein, photolyase, and
at least under these conditions seems to inhibit DNA repair. Previously
it was reported that a CPD is a strong block to Escherichia coli RNA polymerase and that the stalled polymerase inhibits the
recognition of the CPD by components of the UvrABC repair
system(6) . Thus, the mechanism of transcription-coupled repair
must include a process by which the encumbrance of the arrested
polymerase at the site of damage is overcome. On the basis of studies
using cell-free extracts from E. coli, Selby and Sancar (7) proposed that the arrested polymerase is removed from the
DNA template by a transcription repair coupling factor. In order to
resume transcription after repair, an RNA polymerase would have to
reinitiate at the promoter. It remains unclear whether a similar
mechanism is utilized by eukaryotes or whether the polymerase is moved
away from the site of damage without dissociating from the
template(8, 9) . Once the template is repaired, the
arrested polymerase could resume elongation without releasing the
incomplete transcript.
The role of RNA pol II in
transcription-coupled repair can be examined more closely by comparing
the extent of RNA pol II blockage by different DNA lesions with the
extent of transcription-coupled repair of these lesions. DNA lesions
formed by the potent carcinogen N-2-acetylaminofluorene
provide good candidate lesions for such a study. AAF is activated in vivo and binds to DNA predominantly at the C-8 position of
guanine. The major adduct observed is the deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene adduct, but AAF also
forms the N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct.
Although the AAF adduct differs from the AF adduct only by the presence
of an acetyl group, the two lesions have quite distinct structural
effects on DNA. The fluorene moiety of the AAF adduct is inserted
between base pairs within the helix and the guanine switches from the
anti to the syn conformation, leading to a great deal of helix
distortion(10, 11) . In contrast, the fluorene moiety
of the AF adduct remains outside of the helix, and consequently, the AF
adduct does not induce much distortion(12) . These structural
differences are reflected by the types of mutations induced by each
lesion in E. coli. AAF adducts induce primarily frameshift
mutations(13, 14) , whereas AF adducts induce mainly
GC to T
A base pair substitutions(15) .
Interestingly, AAF-treated plasmids replicated in human cells lead to
both types of mutations(16, 17) .
The structural differences between these two adducts are also reflected by the effect each has on the action of various polymerases. An AAF adduct is a much stronger block than an AF adduct to the progression of E. coli DNA polymerase I(18, 19) , T7 DNA polymerase (19, 20, 21) , and T7 RNA polymerase(22) . Both adducts, however, are strong blocks to transcription by RNA pol III from Xenopus laevis(23) .
Both AF and AAF adducts are repaired by the nucleotide excision repair pathway(24, 25, 26) . Interestingly, Tang et al.(27) reported no difference in the rate of repair of AF adducts in a DNA fragment within the transcriptionally active DHFR gene compared with a transcriptionally silent fragment downstream of the gene in Chinese hamster ovary cells. These results suggest that AF adducts are not repaired in a transcription-coupled manner, and taken together with the evidence for bypass of AF-dG adducts by T7 RNA polymerase, it is consistent with our model that an arrested RNA pol II directs repair to the transcribed strand of active genes. A direct correlation between the lack of transcription-coupled repair of an AF adduct and its transcriptional bypass cannot be made, however, until the effects of an AF adduct specifically on a eukaryotic RNA pol II are determined.
In this study, we constructed DNA templates containing AF-dG and AAF-dG adducts located at specific sites downstream from a strong eukaryotic promoter. We found that an AF adduct located on the transcribed strand was a weak block, whereas an AAF adduct on the transcribed strand was a strong block to RNA pol II elongation. In contrast to our previous results using DNA templates containing a CPD, the transcription elongation factor SII did not induce transcript cleavage by RNA pol II stopped by an AAF adduct, suggesting that transcription was terminated. We also found that either an AF or an AAF adduct situated on the nontranscribed strand enhanced a sequence-specific pause site for RNA pol II.
Figure 1: DNA templates. Plasmids pAdCla1, pAdCla2, and pAdCla3 were constructed as described in the text. Gapped duplexes were used to insert oligonucleotides of the sequence 5`-ACTCATCG*ATACTC-3` (shown in bold) into either the transcribed (pAdCla1) or nontranscribed (pAdCla2 and pAdCla3) strand downstream of the adenovirus major late promoter (MLP). G* indicates the position of the adducted guanine residue.
Figure 2:
Transcription of templates containing
specifically located AF and AAF DNA adducts. Templates were linearized
with HindIII and transcribed in vitro such that
transcripts were labeled with P as described in the text.
Elongation was allowed to proceed for 15 min after the addition of NTPs
to the reaction mixture. RNA was then isolated and electrophoresed
through a polyacrylamide gel. Templates containing an unadducted dG,
AF-dG, or AAF-dG are indicated by G, AF, or AAF, respectively. Oligonucleotides were ligated into either
the transcribed (T) or nontranscribed (N) strand. The
position of 370-nucleotide full-length run-off transcripts is indicated
by RO. Transcripts arrested at the damage site or 15 bp
downstream of the damage site are indicated by G* and 185, respectively. Lane 1 contains
X-174
DNA/HaeIII molecular weight markers, whose lengths in base
pairs are given to the left of the lane. Lane 2 shows
transcription of pAdCla1 linearized with ClaI.
Figure 3:
Time course of transcription. Templates
containing either AF-dG (lanes 2-5) or an unadducted dG (lanes 6-9) on the transcribed strand were transcribed in vitro. Samples were removed from each reaction mixture
after 5, 10, 15, or 30 min as indicated, and transcripts were analyzed
as described in the legend to Fig. 2. Lane 1,
X-174 DNA/HaeIII molecular weight markers. RO,
full-length run-off transcripts; G*, 170-nucleotide paused
transcript.
Figure 4: SII does not induce transcript cleavage by RNA pol II arrested by AF or AAF adducts. Transcription complexes arrested by AF-dG (lanes 1-3) or AAF-dG (lanes 4-6) were purified and incubated with SII and NTPs for 60 min as indicated. Transcripts were then analyzed as described in the legend to Fig. 2. RO, full-length run-off transcripts; G*, 170-nucleotide arrested transcript.
Figure 5:
Time course of transcription of templates
containing an AAF adduct on the nontranscribed strand. Templates
containing either an unadducted dG (lanes 2-5) or AAF-dG (lanes 6-9) located on the nontranscribed strand were
transcribed in vitro as in Fig. 2. Samples were removed
from each reaction mixture at the times indicated. Lane 1,
X-174 DNA/HaeIII molecular weight markers. RO,
full-length run-off transcripts; 185 nt, paused
transcript.
Figure 6:
SII induces nascent transcript cleavage by
RNA pol II arrested by an AAF-dF on the nontranscribed strand.
Transcription complexes arrested by AAF-dG on the nontranscribed strand
were purified and incubated with SII and NTPs for 30 min as indicated. RO, full-length run-off transcripts; 185, paused
transcript. X-174 DNA/HaeIII molecular weight markers
not shown.
Figure 7:
Transcription of pAdCla3 templates.
Templates containing either an AF-dG (lane 2) or an AAF-dG (lane 3) were linearized with PvuII and transcribed in vitro as in Fig. 2. Lanes 4-6 show
370-, 170-, and 194-nucleotide run-off transcripts from transcription
of pAdCla3 linearized by PvuII (P), ClaI (C), and HindIII (H), respectively. Lane
1, X-174 DNA/HaeIII molecular weight
markers.
We have examined the effects of AF and AAF DNA adducts on transcript elongation by rat liver RNA pol II. An AF-dG located on the transcribed strand of the template was only a weak block to the polymerase, whereas an AAF-dG located on the transcribed strand was an absolute block to transcriptional elongation (Fig. 2). Similar results have been reported for the effects of these lesions on transcription by T7 RNA polymerase (22) and on replication by DNA polymerases(18, 19, 20, 21) . The translesional bypass of an AF adduct by RNA pol II we observed contrast with results reported for X. laevis RNA pol III, which was blocked by the presence of an AF adduct(23) . This difference is most likely explained by differences in the structure and function of the relatively termination-prone RNA pol III compared with RNA pol II. The nucleotide sequence context of the lesion in the respective systems, however, could also account for the differences in the results.
The different effects that these two adducts have on transcription by RNA pol II are most likely due to the different structural effects each adduct has on DNA. AAF-dG induces local denaturation of double-stranded DNA described as the insertion-denaturation model, whereas AF-dG does not appear to induce denaturation described as the outside-binding model(10, 11, 12) . The guanine of AAF-dG remains in the syn conformation even in single-stranded DNA and may therefore be a noncoding nucleotide for RNA pol II, thereby blocking elongation. Noncoding regions on the transcribed strand are bypassed by T7 RNA polymerase(33) , however, and it is likely that the bulky nature of the AAF adduct also plays a role in transcriptional arrest. Intrinsic arrest sites for RNA pol II are often associated with bends in the DNA helix(34) , and it is likely that the AAF-induced helix distortion combined with the noncoding nature of the adduct contributes to the termination of RNA pol II by AAF-dG. An AF adduct induces little DNA denaturation or helix distortion; therefore it comes as no surprise that AF-dG is not a strong block to chain elongation by RNA pol II.
We have shown that RNA pol II pauses at an
AF-dG but that given time, the polymerase can bypass the adduct (Fig. 3). A similar effect has been reported for replication of
AF-adducted DNA templates by T7 DNA polymerase(21) . In this
case, pausing by the polymerase at AF-dG was ascribed solely to a
decrease in the rate of incorporation of a nucleotide opposite the
adducted nucleotide. It is likely that a decrease in the rate of
incorporation of a ribonucleotide opposite AF-dG is responsible for the
pausing by RNA pol II. Interestingly, although the rate of
incorporation of dCMP opposite AF-dG by T7 DNA polymerase was faster
than the rate of incorporation of dAMP, the latter rate was significant
and suggests a mechanism for the induction of GC
T
A
base pair substitution mutations by AF adducts in
vivo(21) . It is unknown whether the rate of incorporation
of AMP by RNA pol II opposite the lesion is significant.
The transcription elongation factor SII induces transcript cleavage by RNA pol II arrested by a CPD located on the template strand(5) . The fact that cleaved transcripts can be re-elongated up to the site of the CPD demonstrates that the arrested complex is stable and competent to resume transcription. Although an AAF adduct on the transcribed strand is also an absolute block to RNA pol II, SII did not induce transcript cleavage by RNA pol II (Fig. 4). This differential response to SII may be explained by differences in the stability of the arrested complexes. It has been previously shown that when RNA pol III is arrested by an AAF adduct, the polymerase dissociates from the DNA template releasing the nascent transcript(23) . If an analogous response occurs with RNA pol II, SII could not induce transcript cleavage because the ternary transcription complex has been disrupted and transcription effectively terminated. It has been suggested that the stability of an elongation complex is dependent upon the stability of the RNA-DNA duplex(35) ; however, the stability of the RNA pol II elongation complex also appears to be dependent upon DNA binding domains at both the downstream and upstream edges of the transcribing polymerase and on an RNA binding domain that is thought to hold the nascent transcript(36) . It seems likely that the decreased stability of the arrested complex is due to steric interference by the bulky AAF adduct, more so than the interference caused by a CPD. Alternatively, the AAF adduct may not affect the stability of the arrested complex but might specifically inhibit the cleavage reaction, perhaps by influencing the conformation or recognition of RNA pol II by SII. Current studies using footprinting analysis are designed to determine directly whether the polymerase has been dissociated from the DNA at an AAF adduct.
Both AF-dG and AAF-dG when located on the
nontranscribed strand impeded elongation by RNA pol II (Fig. 2).
The arrest site was located 15 bp downstream from the site of the
lesions and was coincident with a pause site for the polymerase. These
blocked complexes were stable and could undergo SII-induced transcript
cleavage (Fig. 6). Removal of the sequence-specific pause site
abolished the adduct-specific blockage, suggesting that the adducts
simply enhance RNA pol II pausing at this site. Indeed, the time course
of transcription study confirmed that an AAF lesion increased the pause
time at this site (Fig. 5). It has been shown that increasing
the dwell time of an elongating transcription complex at a particular
nucleotide sequence can induce a switch from a paused complex in which
the polymerase remains elongation competent to an arrested complex in
which accessory factors are required for elongation to
resume(29, 37) . It is likely that AF-dG and AAF-dG
also induce a shift to polymerase arrest at the intrinsic pause site.
The footprint of an arrested elongation complex covers
30 bp
centered over the transcript arrest site(38) , suggesting that
the AF and AAF adducts are located at the upstream edge of the complex
when the polymerase is paused. The lesions may increase the dwell time
of RNA pol II at this site by interfering with the upstream DNA binding
site of the polymerase impeding translocation past this site. It is
noteworthy that the AAF adduct, which induces a ``hinge
point'' on the DNA helix(10, 39) , has a stronger
effect on this intrinsic pause site compared with the less distorting
AF adduct.
The translesional bypass of AF-dG by RNA pol II is consistent with a model for the mechanism for transcription-coupled repair in which the arrested polymerase directs repair enzymes to DNA lesions located on the transcribed strand of active genes(1, 4) . Repair of AF adducts does not appear to be coupled to transcription. AF adducts located within the active DHFR gene of Chinese hamster ovary cells were repaired at a rate similar to that for adducts located in a transcriptionally silent region downstream of the gene(27) . Although RNA pol II pauses at an AF adduct, the period of pausing may not be long enough to attract the transcription-repair coupling factor(s) that are needed to recruit repair proteins to the arrested polymerase. Similarly, RNA pol II paused at AF-dG does not require SII to bypass the adduct. Even though an AAF-dG is a strong block to RNA pol II, it also may not be a good substrate for transcription-coupled repair if it provokes polymerase release associated with transcriptional termination. It remains unclear whether AAF adducts located on transcribed strands are preferentially repaired because in many eukaryotic cells the AAF-dG is rapidly deacetylated to form AF-dG(40) . In contrast to the results for AF and AAF adducts, a CPD is a strong block to RNA pol II, and the resulting stable arrested complex is probably recognized by a transcription-repair coupling factor leading to the preferential repair of these lesions.
The transcriptional arrest induced by AF and AAF adducts located on the nontranscribed strand reveals the importance of sequence context on polymerase blockage and in turn transcription-coupled DNA repair. If sequence context plays an important role in RNA pol II bypass of a particular DNA lesion, then it must play an important role in transcription-coupled repair of that lesion. Sequence-specific heterogeneity in the rates of repair of CPDs have been reported for both the p53 gene (41) and the PGK1 gene (42) of human cells. Sites where CPDs were shown to be repaired at a slow rate were also shown to be hot spots for mutation, including some sites implicated in tumorigenesis(41) . The slow repair rate of CPDs on the transcribed strand may reflect efficient bypass by RNA pol II in a specific sequence context.