©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of Aminofluorene and Acetylaminofluorene DNA Adducts on Transcriptional Elongation by RNA Polymerase II (*)

(Received for publication, October 30, 1995)

Brian A. Donahue (1) Robert P. P. Fuchs (2) Daniel Reines (3) Philip C. Hanawalt (1)(§)

From the  (1)Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, (2)UPR 9003, Cancérogenèse et Mutagenèse Moléculaire et Structurale, Centre Nationale de la Recherche Scientifique, Ecole Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France, and the (3)Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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 GbulletC to TbulletA 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.


MATERIALS AND METHODS

Proteins and Reagents

RNA pol II, transcription initiation factors, and SII were purified from rat liver or recombinant sources as described previously(28, 29) . D44 IgG anti-RNA antibodies (30) were purified from ascites fluid as described previously(28) . Highly purified NTPs were purchased from Pharmacia, and formalin-fixed Staphylococcus aureus was from Life Technologies, Inc. Radiolabeled nucleotides were purchased from Amersham Corp.

Plasmids

Construction of plasmids pAdBam and pAdSma1 has been described(5) . To construct plasmids to receive AF- and AAF-adducted oligonucleotides, 24-base oligonucleotides with the sequences 5`-GATCCCGAGTATCGATGAGTGGG-3` and 5`-GATCCCCACTCATCGATACTCGGG-3` were annealed and ligated into the BamHI site of pAdBam in one orientation to yield pAdCla1 and in the opposite orientation to yield pAdCla2 (Fig. 1). Both plasmids are identical to pAdSma1 except that the SmaI recognition sequence is interrupted by 14-bp fragments containing the recognition sequence of ClaI. Plasmid pAdSma2 was constructed by replacing the 215-bp BamHI-SphI fragment of pAdBam with a duplex oligonucleotide made by annealing the oligonucleotide 5`-GATCCCCGGGCATG-3` with 5`-CCCGGG-3`. Plasmid pAdCla3 was constructed by replacing the 215-bp BamHI-SphI fragment of pAdBam with the oligonucleotides 5`-GATCCCCACTCATCGATACTCGGGCATG-3` and 5`-CCCGAGTATCGATGAGTGGG-3`. Plasmid pAdCla3 is identical to pAdSma2 except that the SmaI recognition sequence is interrupted by the same 14-bp fragment that interrupts the SmaI recognition sequence of pAdCla2.


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.



Synthesis of Oligonucleotides Containing a Single AF or AAF Adduct

Fourteen-base oligonucleotides of the sequence 5`-ATCCATCGATACTC-3` were prepared by automated DNA synthesis and purified from a polyacrylamide gel. A portion of the oligonucleotides was treated with N-acetoxy-N-2-acetylaminofluorene to form an AAF adduct at the C-8 position of the single G in the oligonucleotide as described(21) . A portion of the 14-AAF oligonucleotides was incubated with 1 N NaOH and 0.25 M beta-mercaptoethanol to deacetylate the AAF adduct, thereby forming an AF adduct on the G residue(31) . All three oligonucleotides were purified extensively by HPLC and characterized by their UV absorption spectra. The fact that the 14-AAF and 14-AF eluted at different positions in the HPLC chromatogram showed that the deacetylation reaction went to completion. Oligonucleotides containing an AF adduct were stored in TE buffer (10 mM Tris, pH 8, 1 mM EDTA, pH 8) with 10 mM beta-mercaptoethanol, whereas the 14-dG and 14-AAF oligonucleotides were stored in TE alone. The presence and the position of the AAF and AF adducts were verified by their ability to block the 3` 5` exonucleolytic activity of T4 DNA polymerase and by the cleavage pattern obtained upon heating the adducted oligonucleotides in the presence of piperidine (data not shown).

Insertion of Adducted Oligonucleotides into Plasmids

DNA templates containing a single AF or AAF adduct on either the transcribed or nontranscribed strand were constructed by utilizing gapped duplexes (GD) as described previously(5) . Oligonucleotides were phosphorylated by T4 polynucleotide kinase and inserted into GD using T4 DNA ligase. DNA ligation products were purified from an agarose gel containing 0.5 µg/ml ethidium bromide. Under these conditions, covalently closed circular DNA migrates as supercoiled DNA and can be resolved from nicked plasmids that resulted from incomplete ligation of the oligonucleotides into the GD. Plasmids pAdSma1 and pAdCla1 were used to make GD in which the adducted oligonucleotides ligated into the transcribed strand (Fig. 1). Plasmids pAdSma1 and pAdCla2 or pAdSma2 and pAdCla3 were used to make GD in which the adducted oligonucleotides ligated into the nontranscribed strand.

Transcription Reactions and Nascent Transcript Cleavage

Transcription reactions were performed as described previously(28) . Briefly, 50-100 ng of DNA templates were linearized with HindIII and incubated at 28 °C with rat liver protein fractions D (2 µg, containing TFIID and TFIIH), B` (1 µg, TFIIF/TFIIE), recombinant rat TFIIB (3 ng), and rat liver RNA pol II (0.5 µg) to form preinitiation complexes. Nascent transcripts were radiolabeled at their 5` ends by the addition of ATP, UTP, and 40 µCi of [alpha-P]CTP. Elongation proceeds until position 15, where the first GTP was required for incorporation. Heparin was added to prevent further initiation, and then 800 µM of each NTP was added to allow elongation to continue, typically for 15 min. Reactions were stopped with SDS and proteinase K and the RNA was precipitated twice in EtOH to remove unincorporated nucleotides. RNA was resuspended in formamide loading dye, heat denatured, and electrophoresed through a polyacrylamide (19:1 acrylamide/bisacrylamide) gel in TBE (89 mM Tris, 89 mM boric acid, 1 mM EDTA, pH 8) with 8.3 M urea. Gels were dried and autoradiographed using intensifying screens. For transcript cleavage experiments, elongation complexes were precipitated with D44 anti-RNA antibodies and formalin-fixed S. aureus and then washed in reaction buffer [20 mM TrisbulletHCl, pH 7.9, 3 mM HEPESbulletNaOH, pH 7.9, 60 mM KCl, 0.5 mM EDTA, 2 mM dithiothreitol, 0.2 mg/ml acetylated bovine serum albumin, 3% (v/v) glycerol, and 2.2% (w/v) polyvinyl alcohol]. Purified elongation complexes were incubated with SII and NTPs in the presence of 7 mM MgCl(2) as described in each experiment. Reactions were stopped, and the RNA was analyzed as described above.


RESULTS

Construction of Templates Containing AF and AAF Adducts at Defined Sites

To study the effects of AF-dG and AAF-dG on transcription elongation by RNA pol II, DNA templates were constructed in which the adducts were located at specific sites downstream of the major late promoter of adenovirus (Fig. 1). Adducts on the transcribed strand were located 170 bp from the transcriptional start site, and adducts on the nontranscribed strand were located 171 bp from the transcriptional start site. The presence of the adducts in these templates was confirmed by their resistance to cleavage by ClaI (data not shown). Templates were linearized by HindIII prior to transcription in order to obtain a distinct 377-nucleotide full-length run-off transcript in the absence of polymerase arrest.

AF and AAF DNA Adducts Block Elongation by RNA pol II to Different Extents

Adducted DNA templates were used in an in vitro transcription assay to determine the extent of RNA pol II blockage by each adduct. An AF adduct located on the transcribed strand was a weak block to RNA pol II as shown by the large proportion of full-length run-off transcripts compared with truncated transcripts (Fig. 2). An AAF adduct located on the transcribed strand, on the other hand, was a complete block to RNA pol II. All transcripts were 170 nucleotides in length, suggesting that the polymerase transcribes the DNA up to but not past the adduct. The length of the truncated transcripts was confirmed by comparison with the 171 nucleotide run-off transcript from ClaI-digested pAdCla1. Interestingly, transcripts were blocked 15 bp downstream from either an AF or an AAF adduct situated on the nontranscribed strand. As was the case for the adducts on the transcribed strand, an AAF adduct on the nontranscribed strand was a stronger block to polymerase than was an AF adduct (Fig. 2, lanes 6 and 8). Templates prepared with unadducted oligonucleotides on either strand yielded full-length run-off transcripts, indicating that the truncated transcripts were due specifically to the presence of the AF and AAF adducts on the DNA templates.


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.



RNA pol II Pauses at an AF Adduct Located on the Transcribed Strand

The small amount of short transcripts observed with templates containing AF-dG on the transcribed strand could be either the result of a small population of polymerase molecules being completely arrested by the adduct or could indicate that the adduct generates a pause site for RNA pol II and that given enough time, the polymerase can bypass this adduct. To test these possibilities, the progression of the transcription complex past the AF adduct was examined at various times by removing samples from the reaction mixture 5, 10, 15, and 30 min after the addition of NTPs. Under the conditions of the transcription assay, RNA pol II did not reinitiate at the promoter; hence, only one round of transcription was observed. As shown in Fig. 3(lanes 6-9) incomplete transcripts of many sizes could be seen from undamaged templates after 5 min, but by 10 min nearly all the transcripts were full-length. On the other hand, only incomplete transcripts of one size were seen from templates containing the AF adduct on the transcribed strand after both 5 and 10 min (Fig. 3, lanes 2 and 3). These transcripts were 170 nucleotides in length, suggesting that all polymerase molecules transcribed up to but then paused at the site of the lesion. The polymerases eventually continued past the AF adducts, however, because by 30 min virtually all transcripts were full-length (Fig. 3, lane 5). Thus, AF-dG in this particular nucleotide sequence context is a pause site for RNA pol II but not a complete block; the polymerase eventually bypasses the AF adduct in the absence of repair.


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.



SII Does Not Induce Nascent Transcript Cleavage by RNA pol II Arrested by an AF or an AAF Adduct

The transcription elongation factor SII facilitates read-through past certain types of transcriptional pause sites often associated with bends in the DNA helix(32) . Integral to this process is the endonucleolytic cleavage of an oligonucleotide from the 3` end of the nascent transcript by RNA pol II before elongation can resume. Presumably, this cleavage allows the polymerase another chance to transcribe through an arrest site. Previously, we showed that SII induces transcript cleavage by RNA pol II arrested by a cyclobutane pyrimidine dimer(5) . To determine whether this process occurs when RNA pol II is arrested by either AF-dG or AAF-dG on the transcribed strand, DNA templates were first transcribed in vitro, and elongation complexes were immunopurified from the reaction mixture and incubated with SII. The vast majority of the truncated transcripts remained 170 nucleotides in length (Fig. 4, lanes 2 and 5), indicating that SII did not induce transcript cleavage by RNA pol II blocked by either an AF or an AAF adduct located on the transcribed strand. The result for AF-dG was not surprising in light of the fairly efficient bypass of this lesion shown in Fig. 3. The lack of transcript cleavage by the polymerase arrested by an AAF adduct, however, is in direct contrast to the case for RNA pol II arrested by a CPD. It is likely that the complex blocked by AAF-dG adduct is unstable and dissociates from the DNA, thereby terminating transcription and precluding SII-induced transcript cleavage.


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.



An AAF Adduct on the Nontranscribed Strand Enhances an Intrinsic Pause Site

A clue to the nature of the polymerase arrest by AF and AAF adducts located on the nontranscribed strand is given in the time course of transcription of unadducted templates by RNA pol II shown in Fig. 3. The most predominant incomplete transcript observed at 5 min migrates as 185 nucleotides, approximately the same length as the truncated transcripts resulting from transcription of templates containing either AF-dG or AAF-dG on the nontranscribed strand (Fig. 2, lanes 6 and 8). To determine more precisely if the adduct-related arrest site coincides with this intrinsic pause site, a time course of transcription was examined for DNA templates in which either unmodified or AAF adducted oligonucleotides had been ligated into the nontranscribed strand (Fig. 5). The progression of transcript elongation on unadducted templates (Fig. 5, lanes 2-5) resembled the results presented in Fig. 3. After 5 min a number of incomplete transcripts including a 185-nucleotide species were seen, but by 15 min nearly all transcripts were full-length, the one exception being a small amount of 185-nucleotide transcript. For the templates containing AAF adducts (Fig. 5, lanes 6-9) at 5 min most transcripts were incomplete. Furthermore, even after 15 min the greatest population of transcripts was 185 nucleotides, suggesting that AAF-dG on the nontranscribed strand increases the time the polymerase is arrested at this site. We have determined that RNA pol II remains arrested at this site for at least 1 h (data not shown).


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.



SII Induces Nascent Transcript Cleavage by RNA pol II Arrested by AAF-dG Located on the Nontranscribed Strand

In an effort to determine if SII induces transcript cleavage by RNA pol II arrested by an AAF adduct on the nontranscribed strand, arrested complexes were purified and incubated with SII for 30 min. Unlike the case in which the adducts were located on the transcribed strand, SII did induce transcript cleavage by RNA pol II arrested by an AAF adduct on the nontranscribed strand (Fig. 6). Transcripts were shortened by approximately 5, 10, and 25 nucleotides from the 3` end (Fig. 6, lane 3). Furthermore, the addition of NTPs after the induction of transcript cleavage resulted in an increase in the amount of run-off transcripts (Fig. 6, lane 4), suggesting that SII facilitates bypass of the pause site when the AAF lesion is located on the nontranscribed strand. These results also show that an AAF-dG itself does not necessarily terminate transcription but that the particular strand where the adduct is located also plays a role. Similar results of SII-induced transcript cleavage were observed for templates containing AF-dG on the nontranscribed strand (data not shown).


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.



Removal of the Intrinsic Pause Site of pAdCla2 Eliminates RNA pol II Arrest by AF-dG and AAF-dG Located on the Nontranscribed Strand

The sequence specificity of the transcription arrest by the adducts on the nontranscribed strand was examined by changing the sequence downstream of the adduct site. A 215-bp fragment between the ClaI and HindIII sites and including the pause site of plasmid pAdCla2 was removed creating plasmid pAdCla3 (Fig. 1). This plasmid was linearized with PvuII to yield a 370-nucleotide full-length run-off transcript. As seen in Fig. 7, nearly all transcripts from templates containing either lesion on the nontranscribed strand of plasmid pAdCla3 were full-length. A small amount of truncated transcripts was seen for both adducted templates (Fig. 7, lanes 2 and 3) that map to the ligation sites of the oligonucleotides, thereby suggesting that a single-stranded nick on the nontranscribed strand may block transcriptional elongation. In addition, a small amount of 185-nucleotide transcript was seen for the AAF-dG template but not nearly to the same extent as was seen for AAF-adducted pAdCla2. For comparison of the lengths of transcripts, unadducted pAdCla3 digested with PvuII, ClaI, and HindIII were used in the transcription assay to yield 370-, 170-, and 187-nucleotide run-off transcripts (Fig. 7, lanes 4-6). Based on these results, we suggest that the 215-bp fragment contains an intrinsic pause site and that removal of this site eliminates the RNA pol II arrest induced by AF-dG or AAF-dG located on the nontranscribed strand.


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.




DISCUSSION

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 GbulletC TbulletA 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.


FOOTNOTES

*
This work was supported by Outstanding Investigator Grant CA 44349 from the National Cancer Institute (to P. C. H.) and Postdoctoral Fellowship PF-3594 from the American Cancer Society (to B. A. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 415-723-2424; Fax: 415-725-1848.

(^1)
The abbreviations used are: RNA pol II, RNA polymerase II; AF, N-2-aminofluorene; AF-dG, N-(deoxyguanosin-8-yl)-2-aminofluorene; AAF, N-2-acetylaminofluorene; AAF-dG, N-(deoxyguanosin-8-yl)-2-acetylaminofluorene; CPD, cyclobutane pyrimidine dimer; GD, gapped duplexes; bp, base pair(s); HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

We are indebted to Joyce Hunt and John Mote, Jr., for expert technical assistance. We thank C. Allen Smith and Ann Ganesan for helpful discussions and for critical reading of this manuscript.


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