Recovery of RNA synthesis from the DHFR gene following UV-irradiation precedes the removal of photolesions from the transcribed strand

Mats Ljungman

Department of Radiation Oncology, Division of Cancer Biology, University of Michigan Comprehensive Cancer Center, 4306 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0936, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is thought that recovery of RNA synthesis following UV-irradiation is closely related to the removal of UV-induced lesions from the transcribed strand of active genes. To test this hypothesis, nascent RNA synthesis from three different locations within the DHFR gene in CHO cells was assessed following exposure to UV light (254 nm). Using both in vivo RNA labeling as well as the nuclear run-on technique, it was found that RNA synthesis from the middle and the 3'-end of the gene was inhibited within 20 min by ~30 and 70%, respectively, while RNA synthesis from the 5'-end of the DHFR gene was enhanced. RNA synthesis from the middle portion of the gene fully recovered within 30–45 min of post-UV incubation, while recovery was slower from the 3'-end of the gene. Compared with previously published data for the kinetics of removal of UV-induced DNA lesions from the 5'-half of the DHFR gene in these cells, it is concluded that RNA synthesis resumed significantly faster in this region than could be accounted for by the removal of photolesions from the transcribed strand. Thus, although RNA synthesis was initially inhibited by UV-induced photolesions, the results suggest that RNA polymerase II was able to bypass these lesions prior to their removal.

Abbreviations: CPD, cyclobutane pyrimidine dimer; DHFR, dihydrofolate reductase gene; hMT, human metallothionein gene; TCR, transcription-coupled repair.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA is constantly being challenged by damaging agents from both endogenous sources and from the environment. DNA repair enzymes have evolved to minimize the risk of converting DNA damage into permanent mutations during replication (1). Certain DNA lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts block the elongation of RNA polymerse II (28). RNA polymerases blocked at CPDs in the transcribed strand have been suggested to trigger both the p53 response (912) and the induction of apoptosis in human cells (10,12,13). Thus, it is of great importance for the cell to quickly remove transcription-blocking lesions from the transcribed strand, not only for the resumption of RNA synthesis and the elimination of mutagenic lesions, but also to avoid the triggering of apoptosis.

The removal of UV-induced CPDs from cellular DNA has been shown to be heterogeneous within the genome (1416). CPDs in mammalian cells are removed significantly faster from actively transcribing genes compared with the genome as a whole (17). In particular, the transcribed strands of active genes are selectively repaired (18). It has been suggested that the role of transcription-coupled repair (TCR) is to clear the DNA template of lesions blocking the transcription machinery (1921). Thus, it is expected that the recovery of mRNA synthesis will follow the removal of UV-induced lesions from the transcribed strand.

To test the prediction that the recovery of mRNA synthesis following exposure to UV light is dependent on the removal of photoproducts from the transcribed strand, RNA synthesis from three different locations within the dihydrofolate reductase gene (DHFR) in CHO B11 cells was measured following UV-irradiation and compared with previous data on the removal of CPDs and (6–4) photoproducts from the same gene in the same cells. Both in vivo labeling of nascent RNA and in vitro labeling using the nuclear run-on technique showed a very fast recovery of DHFR mRNA synthesis. Surprisingly, the recovery of mRNA synthesis from the middle portion of the DHFR gene was significantly faster than the previously reported rates for the removal of photoproducts from the transcribed strand of the same region. Thus, it appears that following an initial inhibition, RNA polymerase II is capable of performing translesion RNA synthesis on a UV-damaged DNA template.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture and UV-irradiation
The methotrexate-resistant Chinese hamster cell line CHO K1 B11 [0.5], containing a 50-fold amplification of the DHFR gene (22), was grown in minimal essential medium supplied with 10% dialyzed fetal bovine serum, 2 mM glutamine, 1x non-essential amino acids, 100 IU penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin and 5000 Bq/ml [methyl-3H]thymidine (for nuclear run-ons) or 150 Bq/ml [methyl-14C]thymidine (Amersham) (for in vivo labeling experiments). Methotrexate (Calbiochem, La Jolla, CA) was added to the medium to a final concentration of 0.55 µM to select for cells maintaining amplifications of the DHFR gene. Irradiation of monolayer cells was performed without medium at room temperature using a Westinghouse IL 782-30 germicidal light predominantly emitting 254 nm light at an incidental dose rate of 0.33 J/m2/s (18).

In vivo labeling of nascent RNA
Nascent RNA was labeled in vivo for 15 min at 37°C in a labeling solution containing 200 µCi [5-3H]uridine (Amersham) as previously described (23; Figure 1AGo). Synthesis of nascent RNA was assessed by measuring the amount of 3H retained on a nylon membrane following hybridization of isolated total RNA with DNA probes immobilized on the membrane (Figure 1BGo). The 3H counts obtained from the filters were then normalized to the 14C counts of the input DNA (from the nucleic acid preparations prior to separation of DNA from the RNA sample). The values of relative total RNA synthesis were obtained by the ratio 3H:14C in the isolated total RNA.



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Fig. 1. (A) Experimental procedure. Cells were irradiated with 10 J/m2 of UV light and incubated for different periods of time before nascent RNA was labeled in vivo for 15 min with [3H]uridine. RNA synthesis was also measured using the nuclear run-on technique where transcripts initiated in vivo were extended and labeled for 20 min in vitro with [32P]UTP. Total RNA, labeled either in vivo or in vitro, was then hybridized to immobilized DNA probes. (B) Map over the CHO DHFR gene showing the locations of the probes used for the RNA synthesis studies and the 14 kb KpnI fragment used in previous studies to measure the removal of CPDs (18). The probes were: pZH4, a 1.74 kb EcoRI–HindIII fragment (a gift from G.Spivak, Stanford University); pB6-14, a 5.3 kb BamHI fragment (46); and pB13-7, a 2.1 kb BamHI fragment (46).

 
In vitro nuclear run-on
The nuclear run-on technique was performed as previously described (2325). Following irradiation, the cells were allowed to incubate for different periods of time before nuclei were prepared. RNA transcripts initiated in vivo were then extended in vitro for 20 min at 30°C in 400 µl of labeling solution containing 100 µCi [32P]UTP.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of UV light on in vivo DHFR mRNA synthesis
Using the in vivo labeling technique (Figure 1AGo), it was found that RNA synthesis was differentially affected by UV light within the DHFR gene. Synthesis of RNA from the first 2.5 kb of the DHFR gene was not inhibited by 10 J/m2 UV light (Figure 2Go). This dose of UV light is expected to form ~2.0–2.5 CPDs (17,18,26,27) and ~0.5 (6–4) photoproducts (28,29) in the transcribed strand of the DHFR gene (30 kb) in CHO cells. Thus, the probability of having a transcription-blocking photolesion within the first 2.5 kb of the gene is low. Interestingly, the rate of RNA synthesis in the 5'-region of the gene gradually increased to 150% within 30 min post-UV incubation, suggesting that the rate of initiation of transcription from the DHFR gene was enhanced by exposure to UV light. RNA synthesis from the middle section of the DHFR gene was initially inhibited by ~35%, but returned to control levels within 30 min. Similarly to the data obtained in the 5'-end of the gene, RNA synthesis from the middle part of the gene was markedly enhanced (150% of control) 45 min following UV-irradiation. Finally, RNA synthesis from the last few kilobases of the 30 kb long DHFR gene was initially reduced to ~30% of the control and recovered to ~50–60% within 1 h post-incubation. It can be noted that the kinetics of recovery were significantly slower at the 3'-end compared with the middle portion of the gene. For comparison, the synthesis of total RNA was reduced to ~70% immediately after irradiation with 10 J/m2 with no significant recovery observed within the first hour following exposure.



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Fig. 2. Resumption of total RNA synthesis and RNA synthesis from the DHFR gene in vivo following UV-irradiation. CHO B11 cells were irradiated with 10 J/m2 254 nm light followed by a 0–45 min post-UV incubation period prior to a 15 min [3H]uridine pulse to label nascent RNA in vivo. RNA labeling, isolation and hybridization to specific DNA probes were performed as previously described (23,33). The relative values for total RNA synthesis were obtained by counting a small volume of the isolated total RNA from the treated cells. The value 100% represents the c.p.m. obtained (following background subtraction) from the RNA of unirradiated cells, which were 774, 244 and 650 c.p.m. for the 5'-end, middle and 3'-end, respectively.

 
Effect of UV light on RNA synthesis measured by nuclear run-on
When transcripts, initiated in vivo, were extended in vitro immediately following irradiation, no inhibition of RNA synthesis was observed from any part of the DHFR gene (compare Figure 3AGo, lanes 1 and 2). This was not surprising since inhibition of RNA synthesis most likely does not occur until the elongating RNA polymerase encounters a photolesion in the transcribed strand. Immediately following irradiation, the RNA polymerases are on average many kilobases away from any dimers and, thus, the extension of the RNA transcripts in the nuclear run-on assay would not be expected to be inhibited. However, when the cells were allowed to incubate for 20 min post-UV before the preparation of nuclei, in vitro RNA synthesis from the middle and the 3'-end of the DHFR gene was found to be reduced by 30 and 70%, respectively, while synthesis from the 5'-end of the gene was enhanced by 50–100%. The increased RNA synthesis observed from the promoter-proximal region suggests that initiation of the DHFR gene was enhanced.



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Fig. 3. Resumption of RNA synthesis in vitro from the DHFR gene following 10 J/m2 UV-irradiation measured using the nuclear run-on technique. (A) Composite of autoradiographs of 32P-labeled RNA hybridized to different DNA probes. The rRNA probe pZH8 is a 4.8 kb DNA fragment complementary to most of the 28S rRNA gene of mouse (a gift from G.Spivak, Stanford University). Film exposure of the pZH8 autoradiograph was substantially shorter than for the other probes. (B) The intensity of the DHFR RNA bands in (A) were quantified using a scanning densitometer and expressed relative to unirradiated control cells (lane 1).

 
The recovery of RNA synthesis was found to be much faster in the middle portion of the DHFR gene compared with the 3'-end. Following 40 min post-UV incubation, RNA synthesis from the middle of the DHFR gene had fully recovered while synthesis from the 3'-end was still reduced by ~40% following 60 min of post-UV incubation (Figure 3Go). For comparison, the nuclear run-on results for rRNA are shown. No significant inhibition of rRNA synthesis was observed following exposure to 10 J/m2 UV light using the nuclear run-on assay. Interestingly, the in vivo technique revealed an ~50% inhibition of rRNA synthesis and no recovery within the first 60 min following exposure to 10 J/m2 (data not shown).

Taking the results from the two different experimental approaches together, both methods demonstrate that UV light stimulated initiation of the DHFR gene and that RNA synthesis recovers more rapidly from the middle than from the 3'-end of the gene.

Recovery of RNA synthesis precedes removal of photolesions from the transcribed strand
The experiments described in this study were designed to closely mimic the conditions used in previous studies measuring the removal of UV-induced CPDs from the transcribed strand of the DHFR gene in CHO cells. Therefore, the same cell line, the same cell culturing conditions, the same gene, the same dose of UV light and even the same UV source in the same laboratory was used in this study as was used in a previous study (18). The published studies of the repair of (6–4) photoproducts were performed in a different laboratory and using a dose of 40 J/m2 (28,29).

If recovery of RNA synthesis must be preceded by the removal of UV-induced lesions from the transcribed strand, it is to be expected that the kinetics of photolesion removal and the kinetics of RNA synthesis recovery should be similar. Surprisingly, it was found that the kinetics of recovery of RNA synthesis from the middle portion of the DHFR gene were significantly faster than the kinetics of removal of both CPDs and (6–4) photoproducts from the transcribed strand of the same area of the DHFR gene in CHO B11 cells (18,29; Figure 4Go). Only ~30% of the CPDs (18) and ~15% of the (6–4) photoproducts present in the transcribed strand of the 14 kb KpnI restriction fragment (Figure 1BGo) were removed within the first 2 h post-UV-irradiation. In comparison, RNA synthesis from the middle portion of the DHFR gene had recovered to a level equivalent to the level in the 5'-end of the DHFR gene within <1 h. Thus, recovery of mRNA synthesis from the middle portion of the DHFR gene following UV-irradiation preceded the removal of UV-induced lesions from the transcribed strand of the same region.



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Fig. 4. Comparison of the resumption of RNA synthesis in vivo from the middle portion of the DHFR gene (pB6-14 in Figure 2Go) with the kinetics of removal of either pyrimidine dimers (10 J/m2) or (6–4) photoproducts (40 J/m2) from the 14 kb KpnI fragment spanning the 5'-half of the DHFR gene (Figure 1BGo). The data for the removal of CPDs and (6–4) photoproduct were taken from Mellon et al. (18) and Link et al. (29), respectively.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
UVC-irradiation induces lesions in DNA that efficiently block both RNA and DNA synthesis in cells (30,31). In this study, the rate of nascent RNA synthesis from three different regions within the DHFR gene in CHO cells irradiated with 10 J/m2 were assessed using two independent techniques. The results obtained with either technique show that shortly after UV-irradiation nascent RNA synthesis from the middle and 3'-end of the DHFR gene was inhibited by 30 and 70%, respectively. No inhibition of nascent RNA synthesized from the 5'-end of the DHFR gene was observed. The heterogeneity of the initial inhibition of nascent RNA synthesis within the DHFR gene could be explained by target size theory. The probability of inducing a transcription-blocking lesion increases as the distance between the transcriptional start site and the target sequence increases. Both CPDs (46) and (6–4) photoproducts (7,8) are thought to inhibit elongation of RNA polymerase II. It has been estimated that 2.0–2.5 CPDs (17,18,26,27) and ~0.5 (6–4) photoproducts (28,29) are formed in the transcribed strand of the DHFR gene (30 kb) following irradiation with 10 J/m2 UVC light. The rate of nascent RNA synthesis observed in this study shortly following UV-irradiation is somewhat higher (middle 65 and 3'-end 30%) than predicted from the expected number of lesion-free regions (first 15 kb 30%, entire gene 10%). Our results suggest that ~50% of the UV-induced photolesions in the transcribed strand initially block RNA polymerase II. Alternatively, all lesions in the transcribed strand may initially block elongation of RNA polymerase, but fast repair and/or bypass of some lesions may have already occurred during the first 15–20 min of nascent RNA labeling.

Following post-UV incubation, nascent mRNA synthesis from the middle portion fully recovered within 30–45 min to levels ~50% higher than in unirradiated control cells. A similar enhancement of RNA synthesis was observed from the 5'-region of the DHFR gene. The recovery of nascent RNA synthesis from the 3'-end was significantly slower, suggesting that recovery of RNA synthesis is a sequential event that begins at the 5'-end of the gene and proceeds towards the 3'-end of the gene. This gradient of RNA synthesis recovery may be a reflection of a more efficient removal of photolesions from the 5'-end of genes compared with the 3'-end (26,27,32). A similar pattern of RNA synthesis recovery has been previously observed in the DHFR gene of these cells following removal of the DNA topoisomerase I inhibitor camptothecin (33).

It is thought that the removal of UV-induced DNA lesions by nucleotide excision repair is critical for the recovery of RNA synthesis (31). Surprisingly, nascent RNA synthesis from the 5'-half of the DHFR gene was found to recover much faster than the previously reported removal of UV-induced CPDs and (6–4) photoproducts from the same region of the DHFR gene (18,29; Figure 4Go). While nascent RNA synthesis from the middle portion of the DHFR gene had recovered to levels similar to or even higher than unirradiated control cells within 30–45 min, only ~30% of the pyrimidine dimers and 15% of the (6–4) photoproducts are expected to be removed from the transcribed strand within 2 h following UV-irradiation (18,29). Similar fast recovery of RNA synthesis has previously been reported for the human metallothionein (hMT) gene in UV-irradiated human cells (34). In that study, northern blot analysis of the short-lived hMT mRNA revealed that following an initial decrease in mRNA levels following exposure of cells to 10 J/m2, the steady-state level of hMT mRNA returned to control levels much faster than the UV-induced DNA lesions were removed from the same sequence.

One explanation for the unexpectedly fast recovery of RNA synthesis could be that undamaged alleles may become transcriptionally up-regulated following UV-irradiation. RNA synthesis from the 5'-end is increased to ~150% within 30 min following irradiation, suggesting that UV light stimulates initiation of the DHFR gene. Transcription from the middle region of the DHFR gene initially was inhibited by ~30–35%, but within 45–60 min was transcribed at an increased level indistinguishable from that at the 5'-end. As the levels of transcription from the middle and 5'-regions of the gene are indistinguishable 60 min after UV-irradiation, lesions remaining in the first half of the DHFR gene do not appear to affect transcription. The results support the hypothesis that the initially blocked RNA polymerases are somehow able to bypass photolesions prior to their removal.

If RNA polymerase II is able to bypass photolesions after an initial arrest, how could the phenomena of TCR be reconciled? One possible explanation could be that the transcription-blocking lesions get modified by the transcription machinery to allow translesion RNA synthesis and that these modified lesions are better substrates for the nucleotide excision repair system. This would result in the preferential repair of the transcribed strand without any physical interaction between the RNA polymerase and the repair enzymes. In fact, the intradimer phosphodiester backbone can be cleaved by a cellular endonuclease prior to the removal of the CPDs by the nucleotide excision repair complex (35,36). These modifications tend to accumulate in transcriptionally active DNA sequences and, thus, may function to promote transcription read-through on UV-damaged templates (37). Interestingly, a recent study has found that CPDs with breaks in their phosphodiester backbones are better substrates for the UVrABC endonuclease of Escherichia coli than intact CPDs and, thus, would be expected to be preferentially repaired by the nucleotide excision repair pathway (38). An alternative hypothesis to explain TCR following photolesion bypass is that mismatch repair proteins, which have been shown to be involved in TCR (39,40), may recognize and bind to unpaired bases formed near CPDs by the induction of negative superhelicity in the DNA in the wake of the elongating RNA polymerase II (41,42). The binding of mismatch repair proteins to the DNA surrounding the photolesions may `tag' the damaged sites and perhaps stimulate the assembly of repair complexes.

UV-induced CPDs and (6–4) photoproducts are efficient blocks for the elongation of RNA polymerase II (28). If cells do not rapidly resume RNA synthesis following UV-irradiation, the cells may be induced to undergo apoptosis (10,12,13). Resumption of RNA synthesis could be accomplished either by removing the blocking lesions from the DNA template or by lesion bypass resulting in translesion RNA synthesis. In order to repair the damaged sites, the blocked RNA polymerases must be removed to allow DNA repair enzymes to gain access to the lesions (4,43). Several mechanisms could be considered to remove a stalled RNA polymerase from a damaged site (Figure 5Go). These include a bypass mechanism (this study), `backing up' mechanism, perhaps catalyzed by the transcription factor SII (4,20), a `falling off' mechanism, in which the RNA polymerase may start over at the initiation start site (21), or a degradation mechanism, in which the largest subunit of RNA polymerase becomes ubiquitinated and subsequently degraded in a proteasome-dependent manner (44,45).



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Fig. 5. Resumption of RNA synthesis following blockage of RNA polymerase II at a photolesion can be accomplished either by removal of the blocking lesion or by enabling the polymerase to bypass the photolesion. In order for the repair enzymes to gain access to the photolesion blocking the RNA polymerase, the polymerase must be removed. This could be accomplished by a bypass, a backing up, a falling off or a protein degradation mechanism. The mechanism of photolesion bypass may involve some form of modification of the photolesion.

 
In this study, results were obtained which suggest that UV-irradiated cells may resume RNA synthesis from the DHFR gene prior to removing all the photolesions from the template strand. It is possible that some aspects of nucleotide excision repair and/or TCR are required for this bypass function, since cells with deficiencies in these processes have reduced ability to recover RNA synthesis following UV-irradiation (10,31). However, deficiencies in nucleotide excision repair leads to a more severe degradation of the largest subunit of RNA polymerase following UV-irradiation (45). Thus, the poor recovery of RNA synthesis in repair-deficient cells may be due to the depletion of the inherent transcriptional activity of a cell rather than the inability to bypass photolesions. Future studies will define the underlying mechanisms responsible for this apparent bypass of UV-induced DNA lesions and to determine whether lesion bypass by the initially blocked RNA polymerase may play a role in circumventing triggering of the apoptotic response following UV-irradiation.


    Acknowledgments
 
I would like to thank Dr Philip Hanawalt and the members of his research group for valuable input into this study. I would also like to acknowledge Drs Graciella Spivak, Isabel Mellon, Linus Ho and Lawrence Chasin for their generous gifts of DNA probes and Dr Bruce McKay and Sara Ljungman for critically reading this manuscript. This work was conducted in Dr Hanawalt's laboratory at Stanford University and was supported by an outstanding investigator award to Dr Philip Hanawalt from the National Cancer Institute.


    Notes
 
Email: ljungman{at}umich.edu


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 19, 1998; revised October 20, 1998; accepted November 4, 1998.