©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Variations in Transcription-Repair Coupling in Mouse Cells (*)

(Received for publication, September 1, 1994; and in revised form, November 16, 1994)

Ahmed O. Murad (§) Jeanine de Cock (¶) David Brown Michael J. Smerdon (**)

From the Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-4660

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Formation and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined in three different genes in mouse L cells: 1) a stably integrated insert (called LTL), consisting of a herpes simplex virus thymidine kinase gene (tk) fused to a hormone inducible promotor (LTR); 2) the constitutively expressed proto-oncogene c-abl; and 3) the inactive immunoglobulin J chain gene. Transcription of the tk gene is induced >50-fold by dexamethasone. There is a nonuniform distribution of CPDs in LTL DNA irradiated in vitro, being 4-fold higher in the LTR than in the tk gene, indicating the LTR may be damaged preferentially in irradiated cells. Repair of CPDs occurs efficiently in both strands of LTL and is unaffected by hormone induction of tk gene transcription. Transcription of tk mRNA is very sensitive to UV damage and follows single hit kinetics with UV dose. Furthermore, tk mRNA expression rapidly recovers during repair incubation. Transcription-coupled repair occurs in these cells, however, since only the transcribed strand of c-abl is efficiently repaired of CPDs; the nontranscribed strand as well as both strands of the J chain gene are inefficiently repaired. Thus, repair in the LTL construct may reflect a lack of transcription-coupled repair in either the LTR promotor or the LTL insertion region of chromatin.


INTRODUCTION

It has become increasingly clear that formation and repair of different DNA lesions is not equal across the genome (reviewed in Smerdon, 1991). For example, many transcribed genes are repaired more efficiently than the bulk of the DNA in most eukaryotic cells studied thus far, and this preference extends to the template strand of RNA polymerase II (reviewed in Friedberg et al., 1994). This ``selective repair'' of active genes is very pronounced in rodent cells and may explain earlier observations that these cells are partially deficient in repair of ultraviolet (UV) radiation damage to the genome overall, yet survive as well as human cells that are proficient in overall repair (reviewed in Smith, 1987).

The biological importance of differential repair is implicated in the human genetic disorders Cockayne's syndrome and xeroderma pigmentosum (complementation group C). Cockayne's syndrome cells have lost the ability to preferentially repair transcriptionally active DNA (Venema et al., 1990a; van Hoffen, et al., 1993), while xeroderma pigmentosum (complementation group C) cells have a reduced ability in repairing the bulk of the genome (Venema et al., 1990b; Evans et al., 1993). What is not clear is the mechanism(s) by which this selective repair takes place and how repair processes are linked to transcription and chromatin structure. However, studies on Escherichia coli, demonstrating a repair bias in the transcribed strand and the requirement for the mfd gene product, predict that a transcription-repair coupling factor(s) exists in both prokaryotes and eukaryotes (e.g. see Drapkin et al., 1994).

Transcription-repair coupling may not exist in all cells, however. de Cock and co-workers (de Cock et al., 1991, 1992a) did not observe transcription-coupled repair in a cell line derived from Drosophila melanogaster embryonic cells. These authors found no evidence for preferential repair or strand-specific repair of UV-induced cis-syn cyclobutyl pyrimidine dimers (CPDs) (^1)in the transcriptionally active Gart and Notch genes. They also found efficient repair of CPDs in the inactive white gene and in the genome overall (80-90% CPDs removed in 24 h). Furthermore, no preferential or strand-specific repair of CPDs was observed in the beta(3)-tubulin gene upon induction of transcription (de Cock et al., 1992b). As discussed by these authors, the rate of overall genome repair in D. melanogaster embryonic cells is much higher than in rodent cells, and the lack of transcription-repair coupling may be specific for certain cell types. A similar observation was reported for the cysteine proteinase gene of Dictostelium discoideum (Mauldin et al., 1994). Once again, genomic repair in these cells is very efficient and much higher than in rodent cells (Mauldin et al., 1994). In the present study, we have examined repair of three different types of pol II genes (an inducible foreign gene, a constitutively expressed native gene, and an inactive native gene), to address the question of whether transcription-coupled repair can vary in the same cells.

We have used a mouse cell line (L1.4-3) containing a stably integrated construct (LTL) consisting of the herpes simplex virus thymidine kinase gene (tk) inserted between two long terminal repeats (LTR) from mouse mammary tumor virus (MMTV). The constitutively silent (or near silent) tk gene lacks its own promoter and is transcribed from the glucocorticoid hormone-inducible promoter in the upstream LTR (Zaret and Yamamoto, 1984). Induction of the tk gene is accompanied by changes in chromatin structure, as shown by appearance of broad DNase I sensitivity and specific DNase I-hypersensitive sites (Zaret and Yamamoto, 1984). The broad DNase I sensitivity is enhanced, however, relative to the inactive J chain gene even in the absence of induction (Zaret and Yamamoto, 1984). The MMTV-LTRs are known to contain six precisely positioned nucleosomes, where one of these nucleosomes is positioned over the glucocorticoid response elements (GREs) required for hormonal induction (Richard-Foy and Hager, 1987; Piña et al., 1990b). This nucleosome is important in positioning the sequence for transcriptional induction and is disrupted upon binding of the hormone-receptor complex (Bresnick et al., 1990; Piña et al., 1990b). These features of the LTL construct provide an ideal system to correlate DNA damage and repair with both changes in chromatin structure and transcriptional regulation.


EXPERIMENTAL PROCEDURES

Cell Culture

Mouse cell line L1.4-3, containing the LTL construct stably integrated into the genomic DNA (Zaret and Yamamoto, 1984), was provided by Drs. Kenneth Zaret and Keith Yamamoto (University of California at San Francisco). Cells were grown as monolayers in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with fetal bovine serum (Hyclone), in the absence of phenol red (Picard and Yamamoto, 1987). Cells were usually split 1:4 in medium containing 10% fetal bovine serum, grown for 3-4 days, and the medium replaced with fresh medium containing 5% fetal bovine serum. The cells were grown an additional 2-3 days before being used for an experiment. Forty-five min before UV irradiation, dexamethasone (Sigma, 0.1 mM stock solution in ethanol) was added to a final concentration of 0.1 µM to some plates to induce tk gene expression. For most repair experiments, cells were either prelabeled with 5 nCi/ml [^3H]dThd during rapid growth and density-labeled with 10 µM BrdUrd, 1 µM FdUrd during repair incubation (Smith et al., 1981), or treated with 2 mM hydroxyurea as described previously (Smerdon et al., 1979).

UV Irradiation and DNA Isolation

Cells in monolayer cultures in Petri dishes were placed on ice and washed once with ice-cold TBS (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM KCl). The cells were irradiated in a (desk-top) hood containing a single low pressure mercury lamp (Sylvania, model G 30T8) providing predominantly 254 nm light. The UV flux was measured using either a Black Ray UV meter (Ultra-Violet Products, San Gabriel, CA) or Spectroline DM-254N UV meter (Spectronics Corporation, Westbury, NY). Cells were either harvested immediately after irradiation or incubated for varying repair times and DNA isolated as described in Murad(1991). Briefly, cells were collected by centrifugation (1000 times g), resuspended in nuclei isolation buffer (10 mM Tris, pH 8.0, 0.1 mM CaCl(2), 0.25 M sucrose, 0.5% Triton X-100), and vortexed for 1-2 min. Nuclei were sedimented by centrifugation at 1500 times g and resuspended in STE buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 10 mM NaCl) by gentle vortexing. Sarkosyl was added to a final concentration of 0.5%, and nuclei were digested with 150 µg/ml proteinase K at 42 °C for 8-12 h. The DNA was precipitated twice in ethanol and 2.5 M ammonium acetate, washed with 70% ethanol, and resuspended in TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA).

Aliquots containing 40-60 µg of DNA in 150-200 µl of TE buffer were digested to completion with restriction endonucleases, as determined by electrophoresis on 0.5% agarose mini-gels. DNA was again precipitated and resuspended in TE buffer to a final concentration of 1-1.5 µg/µl. For density-labeled samples, unreplicated DNA was isolated by neutral CsCl gradient centrifugation (Smith et al., 1981; Ausubel et al., 1987).

T4 Endonuclease V Digestion and Gel Electrophoresis

T4 endo V was either purified from E. coli strain AB2480 (see Murad, 1991), containing plasmid pTACdenV provided by Drs. Kristoffer Valerie and Jon de Riel (Temple University, PA), or obtained in purified form from Dr. R. Stephen Lloyd (University of Texas Medical Branch, Galveston, TX). Between 10 and 20 µg of restriction enzyme-digested genomic DNA was aliquoted into microcentrifuge tubes with linearized (unirradiated) plasmid DNA, containing DNA sequences complementary to the probe, as a standard to correct for loading differences between lanes. DNA in T4 endo V digestion buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 100 µg/ml bovine serum albumin) was digested with 1 µl of a 1:10 dilution of T4 endo V preparation for 30 min at 37 °C. The reaction was stopped by addition of concentrated alkaline gel loading buffer (Maniatis et al., 1982). Samples were loaded on 0.6-0.9% alkaline agarose gels (Maniatis et al., 1982) and electrophoresed at room temperature, transferred to nylon membranes (Hybond-N+, Amersham Corp.), and hybridized with strand-specific probes (see below) as described in Murad(1991). Membranes were exposed to (preflashed) x-ray film with intensifying screens and stripped of RNA probes for additional hybridizations (Murad, 1991).

Preparation of Single-stranded Probes

Plasmids containing 1) the entire LTL construct (pLTL1), 2) an incomplete cDNA of the immunoglobulin J chain gene of mouse (pJc21), or 3) a 3.9-kb cDNA clone of the mouse c-abl type IV transcript (pBS-Ty4) were used for generating single strand probes. A 1.5-kb BamHI-NcoI tk gene fragment (see Fig. 1) and 1.0-kb ClaI-PvuII LTR fragment of pLTL1, and a 1.2-kb EcoRI fragment of pJc21, were subcloned into plasmid pBS (Stratagene Cloning Systems, CA) containing T3 and T7 RNA promoters for generation of single strand RNA probes. Strand-specific RNA probes were generated from these plasmids, and plasmid pBS-Ty4, according to the manufacturer's specifications. Strand-specific DNA probes were generated using primer-extension from vector sequences (Ruven et al., 1994).


Figure 1: A, the LTR-tk-LTR (LTL) construction inserted into the genome of mouse L1.4-3 cells (Zaret and Yamamoto, 1984). The LTR regions are derived from an MMTV proviral clone isolated from infected rat cells. The HSV-tk gene lacks its own promoter and is under the control of the LTR promoter. These cells grow in selective (HAT) medium only when treated with dexamethasone (Zaret and Yamamoto, 1984; Murad, 1991). The solid boxes inside the LTR denote the glucocorticoid receptor binding region. The solid lines represent rat DNA flanking the original proviral clone. The top line shows the actual scale (in kb) with the restriction enzyme cut sites for HindIII (H), StuI (S), BamHI (B), and NcoI (N). The wavy arrows under the LTL map indicate the transcripts produced upon induction with dexamethasone, and the shaded boxes denote the regions complementary to the tk probe. B, Northern blot of total cellular RNA with and without treatment with dexamethasone. Antisense RNA probes complementary to the tk gene and the proto-oncogene c-abl (type IV) mRNA were hybridized at the same time. (The weaker band between the 6.5-kb c-abl and 3.8-kb tk bands is only detected with the c-abl probe.) The picture shows a longer exposure for clarity, while the scans next to the picture are from a lower exposure within the film linear range.



RNA Isolation and Northern Blot Analysis

After UV irradiation, the medium (containing 0.1 µM dexamethasone) was added, and cells were incubated for 60-90 min to induce tk gene expression. (For repair experiments, cells were incubated for longer periods (up to 24 h).) Total cellular RNA was isolated from L1.4-3 cells, electrophoresed on formaldehyde agarose gels (1.2%), transferred to nylon membranes, and hybridized with single strand (antisense) probes (Murad, 1991). Membranes were washed and exposed to x-ray films as described above.

Quantitation of Autoradiograms

Film linearity and preflash conditions were determined using published procedures (see Murad, 1991). Quantitation of autoradiograms was performed by scanning with a laser densitometer (LKB model 2222) or by video densitometry using the Visage 60 computer system (Bio Image, MA) and the whole band analysis software for this system. In most cases, band intensities were normalized to the loading standard in each lane, and then to the band intensities of the -T4 endo V samples, to give the fraction of fragments free of CPDs (P(o)). The average number of CPD/fragment was determined from the Poisson equation [-ln(P(o))] (Bohr and Okumoto, 1988).


RESULTS

Induction of tk mRNA by Treatment with Dexamethasone

Fig. 1A shows a map of the LTL construct, as it was inserted into mouse L1.4-3 cells, where two transcripts are generated (Zaret and Yamamoto, 1984). The tk transcript starts in the 5`-LTR and continues to either the polyadenylation site in the tk gene (1.6 kb) or, less frequently, through the partial env gene to the polyadenylation site in the 3`-LTR (3.8 kb; Zaret and Yamamoto, 1984). As shown in Fig. 1B, the 1.6-kb transcript is the major transcript induced (>50-fold) by treatment with 0.1 µM dexamethasone, and little transcription occurs in the absence of dexamethasone (also see Zaret and Yamamoto, 1984). Also shown in Fig. 1B is the 6.5-kb c-abl (type IV) transcript, which is constitutively expressed in these cells, and is unaffected by dexamethasone treatment. (For comparison, the blot shown in Fig. 1B was hybridized with the tk and c-abl probes simultaneously.)

CPD Formation in the LTL Domain

Initially, we measured the CPD yield in each strand of the 13.5-kb HindIII fragment (entire LTL insert) and the 3.7-kb StuI fragment (see Fig. 1A) of LTL using the method of Bohr et al. (1985). This assay measures the fraction of restriction fragments resistant to cutting by T4 endo V, which cleaves DNA strands specifically at CPDs. Cells were harvested immediately after irradiation, and genomic DNA was isolated and digested with either HindIII or StuI. An unirradiated 1.6-kb fragment containing the HSV tk gene was added to each sample, prior to T4 endo V digestion, to correct for loading differences between gel lanes. Samples were then treated (or mock treated) with T4 endo V, run on denaturing agarose gels, transferred to nylon membranes, and hybridized with P-labeled RNA probes containing the tk gene (see Fig. 1A). Fig. 2(top panel) shows sections of an autoradiogram obtained for each digest and probed with RNA complementary to the transcribed strand of the tk gene. Results from quantitation of Southern blots for cells irradiated at different UV doses are shown for both fragments in the bottom panel. The initial frequency of CPDs as a function of UV dose is plotted for both the transcribed (TS) and coding strands (CS) in the presence (dashed lines) or absence (solid lines) of dexamethasone. In the absence of dexamethasone, the average number of CPDs in each strand of the HindIII fragment is about 0.7 CPD/100 kb/J/m^2, as determined from slopes of linear regression fits to the data. These values increased slightly (to about 1.0 CPD/100 kb/J/m^2) for cells irradiated in the presence of dexamethasone. The smaller StuI fragment yielded a higher value (0.9 CPD/100 kb/J/m^2) for each strand in the absence of dexamethasone and was essentially unchanged following treatment with dexamethasone (Fig. 2, C and D).


Figure 2: Southern blot analysis of CPD formation in the LTL insert in mouse L1.4-3 cells. Autoradiograms are for the transcribed strands of the HindIII (A) and StuI (B) digests of genomic DNA isolated from L1.4-3 cells. Cells treated (+) or not(-) with dexamethasone (Dex) were irradiated at 0, 10, or 20 J/m^2 and harvested immediately. In each case, unirradiated loading standard (1.6 kb) was added to each sample prior to digestion with T4 endo V (Loading Control). Southern blots from alkaline agarose gels were probed with P-labeled strand-specific RNA probes complementary to the transcribed strand of the tk gene (see Fig. 1). Lower panels, plots of CPD/fragment as a function of UV dose for both template (C: TS) and coding (D: CS) strands of the StuI (up triangle, ) and HindIII (circle, bullet) fragments. Quantitation of bands resistant to digestion with T4 endo V on autoradiograms (as shown in inset) was performed by video densitometry (see ``Experimental Procedures''). The CPD yield at each UV dose for the 13.5-kb HindIII and 3.7-kb StuI fragments was calculated from the Poisson equation (Bohr and Okumoto, 1988). Dashed lines are for cells treated with dexamethasone (solid symbols), and solid lines are for untreated cells (open symbols). Data from several experiments at different UV doses were plotted as the same symbol in each case. Fits shown are for linear regression analyses of the data having a zero intercept.



The average number of CPD/100 kb/J/m^2 formed in each fragment is shown in Table 1. Our values for CPD formation in the HindIII fragment of the LTL are within the range of values reported for other known sequences of DNA (e.g. see Venema et al., 1990a; Evans et al., 1993). The average number of CPDs in the genome of Chinese hamster ovary cells, as determined from alkaline sucrose gradient profiles, is about 0.7 CPD/100 kb/J/m^2 (Bohr et al., 1985). Taken together, these data indicate that there is no unusual bias for CPD formation when the entire LTL domain is considered. However, we consistently observe frequencies of CPD formation (per unit of DNA) in the 3.7-kb StuI fragment that are about 25% higher than the 13.5-kb HindIII fragment (calculated from data such as that shown in Fig. 2, C and D). Since the HSV-tk gene is GC-rich (36% AT) and the LTR (56% AT) has many long T-tracts, we examined if the HSV-tk gene is less efficient in forming CPDs in plasmid DNA irradiated in vitro. Plasmid DNA was digested with restriction enzymes that liberated fragments from just the tk gene (1.5 kb) and LTR (1.0 kb) regions, and the samples were irradiated at high UV doses (to accurately measure the CPD yield in such short fragments; Bohr and Okumoto, 1988). The LTR fragment forms CPDs about 4-fold more efficiently than the tk gene sequence (Fig. 3). Thus, if most CPDs formed in the StuI fragment in vivo are localized outside the 1.5-kb tk sequence, the average number of CPDs formed in this fragment (per 100 kb/J/m^2) may be much higher than in the longer HindIII fragment.




Figure 3: Comparison of CPD formation in the MMTV-LTR and tk gene sequences irradiated at high UV doses in vitro. Plasmids pBS-tk and pBS-LTR were digested with BamHI and either HindIII (pBS-tk) or EcoRI (pBS-LTR) to liberate a 1.5-kb tk fragment or a 1.0-kb LTR fragment, respectively. Samples in TE buffer (A = 1.0) were irradiated simultaneously in Petri dishes, ethanol precipitated, and resuspended in TE buffer prior to digestion with T4 endo V. Unirradiated, linearized plasmid was added to each sample as a loading control, and samples were run on an alkaline agarose gel. Quantitation of CPD/fragment at each UV dose was performed using a laser densitometer (``Experimental Procedures'') and the data normalized to the fragment lengths in each case. Data are for the LTR (down triangle) and tk gene (bullet) fragments shown in the autoradiogram (inset), which were first normalized to their respective loading control bands. (Some fluctuation in band intensities is due to gel loading variation.) Linear regression analysis yields slopes of 6.4 times 10 and 1.6 times 10 CPD/kb/Jm for the LTR and tk gene fragments, respectively.



This agrees with a computer analysis of the LTL sequence (Murad, 1991), which examines the distribution of potential thymidine dimer (TD) sites, the major type of CPD in random sequence DNA (Patrick and Rahn, 1976). The MMTV-LTR sequence has an unusually high number of potential TD forming sites (e.g. 183 TT sites/kb in the LTR sequence and 25 of these are in runs of four or more), whereas the HSV-tk sequence has a much lower number (92 TT sites/kb and only eight in runs of four or more). This non-uniform distribution of potential TD sites within the LTL sequence becomes even more striking when the effect of adjacent flanking bases is taken into account (Murad, 1991). A similar search for TC pairs, the most prevalent sites for UV-induced) pyrimidine-pyrimidone formation (Franklin and Haseltine, 1986; Mitchell et al., 1990), failed to show any unusual bias in these sequences (110 TC sites/kb in the LTR and 92 TC sites/kb in the tk gene). These results again predict preferential damage by UV radiation in the LTR region in intact mouse L1.4-3 cells.

Effect of UV Damage on tk Gene Transcription

Radiolabeling studies in mouse L cells have shown that total synthesis of RNA is reduced following UV irradiation (Sauerbier, 1976). More recently, it has been shown that a single CPD in a transcribed sequence will inhibit transcription through that sequence (Selby and Sancar, 1990; see also Protic-Sabljic and Kraemer, 1985). Therefore, for short-lived mRNA (i.e.t repair incubation times) Northern analysis yields an indirect assay of the level of transcription blocking UV damage to the DNA of an active gene. Treatment of mouse L1.4-3 cells with the transcription inhibitor actinomycin D yields a half-life of 55 min for the 1.6-kb tk mRNA transcript (Murad, 1991). This indicates that the tk message is in the ``short-lived class'' of mRNAs in mammalian cells (Raghow, 1987) and can be used to monitor repair of the tk gene.

If the inhibition of tk mRNA synthesis results from formation of UV photoproducts within sequences required for tk gene expression, then a plot of tk mRNA level versus UV dose should follow single-hit kinetics (Freifelder, 1982). Therefore, tk mRNA levels in mouse L1.4-3 cells, at 1.5 h after UV irradiation and treatment with dexamethasone, were determined by Northern blot analysis (Fig. 4). Under these conditions, both induction and synthesis of new tk mRNA are required after UV damage to LTL. The tk mRNA levels decline exponentially with UV dose which is typical of one-hit, one-target events, indicating that synthesis of tk mRNA is directly inhibited by UV light. Furthermore, a UV dose of 20 J/m^2 blocks >85% of the tk gene expression (Fig. 4). This result is not predicted for a random distribution of CPDs in LTL, where only 25% of the tk mRNA should be inhibited from direct blockage of RNA polymerase elongation (Fig. 4, dashed line), using the CPD/100 kb/J/m^2 value for the StuI fragment (Table 1). This result suggests that hotspots for CPD formation may exist within sequences essential for tk transcription, and/or other UV photoproducts (e.g.) pyrimidine-pyrimidone dimers) play a major role in affecting tk gene transcription in the LTL.


Figure 4: Northern blot analysis of tk mRNA levels as a function of UV dose. After UV irradiation, dexamethasone was added to a final concentration of 0.1 µM and cells were incubated at 37 °C for 1.5 h. Total RNA was isolated for Northern blot analysis and hybridized to an antisense tk RNA probe. A resulting autoradiogram is shown above the plot. The dashed line approximates the predicted levels of mRNA for a random distribution of CPDs in the 1.6-kb transcribed region of LTL based on values obtained from Southern blot analysis of the whole 13.5-kb HindIII DNA fragment (Table 1).



Repair of Both Strands of the LTL Insert

We initially measured removal of CPDs from both strands of the 13.5-kb HindIII fragment using the Southern blot method and a nick-translated probe to the LTL insert. As shown in Fig. 5A, after 12 J/m^2 UV a fraction of the HindIII fragment (30-40%) remains uncut after T4 endo V treatment, and this fraction is markedly increased following 24 h of repair incubation. Surprisingly, this was observed in both dexamethasone-treated and untreated cells (Fig. 5A). Since some experimental variation was observed in the efficiency of BrdUrd-density labeling in these cells (possibly reflecting differential expression of the tk gene), we wondered if the appearance of efficient repair in the untreated cells was due, in part, to background replicative synthesis. Therefore, we used both the BrdUrd density-shift method, and 2 mM hydroxyurea to suppress replicative synthesis (Murad, 1991), to measure repair of CPDs in mouse L1.4-3 cells. As shown in Fig. 5B, 80% of the fragments are resistant to T4 endo V cutting after the 24-h incubation period in both density-labeled and hydroxyurea-suppressed cells. Thus, the increase in T4 endo V-resistant fragments in uninduced cells cannot be explained by background replicative synthesis.


Figure 5: Removal of CPDs from both strands of LTL in cells exposed to dexamethasone (A) and cells treated with and without hydroxyurea (B). A, autoradiograms for a HindIII digest of genomic DNA isolated from BrdUrd-labeled L1.4-3 cells treated (+) or not(-) with 0.1 µM dexamethasone (Dex) and irradiated with 12 J/m^2 UV light. Density-labeled cells were harvested immediately (0 h) or incubated for 24 h after irradiation and the nonreplicated DNA isolated on CsCl gradients. Southern blots from alkaline agarose gels were hybridized with a nick-translated tk gene fragment of LTL (see Fig. 1). B, top panel, same as A, except mouse L1.4-3 cells were treated (+) or not(-) with 2 mM hydroxyurea (HU) prior to irradiation with 12 J/m^2 UV light. Cells were incubated for 0, 8, or 24 h after irradiation and the nonreplicated DNA was isolated, electrophoresed, blotted, and probed (as for A). B, bottom panel, maximum band intensities of the +T4 endo V samples for each time point shown in the top panel of B, relative to the -T4 endo V samples, for samples treated with (stippled bars) or without (solid bars) hydroxyurea.



To determine if this efficient repair of LTL is reflected by a rapid recovery of tk mRNA expression, the level of full-length tk mRNA was examined after UV irradiation. (This ``in vivo method'' was used for measuring the recovery of tk mRNA transcription, instead of a nuclear run-off assay, since the latter only measures elongation from an already formed RNA polymerase complex and cannot measure effects of UV damage on initiation of the tk transcript.) As shown in Fig. 4, this expression is very sensitive to UV damage, being reduced by >85% after 20 J/m^2 UV light. Northern blot experiments were performed to measure the return of tk mRNA after UV irradiation in cells treated with dexamethasone and hydroxyurea. Fig. 6A shows representative autoradiograms for different times after a UV dose of 20 J/m^2. As can be seen, the level of full-length tk mRNA in irradiated cells lags behind the level observed in unirradiated cells following induction by dexamethasone. Quantitation of autoradiograms, such as those in Fig. 6A, indicates that over 65% of the tk mRNA production in unirradiated cells is recovered following 24 h of repair incubation (Fig. 6B). These data correlate with the efficient repair of CPDs observed in both strands to the LTL (see Fig. 5).


Figure 6: Recovery of tk mRNA in mouse L1.4-3 cells following UV irradiation. A, cells were incubated for varying times in the presence (+) or absence(-) of 0.1 µM dexamethasone following irradiation (or mock irradiation) with 20 J/m^2 UV light. Total cellular RNA was isolated at the indicated times, electrophoresed on formaldehyde-agarose gels, blotted, and hybridized to a P-labeled antisense tk RNA probe (see Fig. 1). B, percent of tk mRNA in UV irradiated cells, as compared to the level in unirradiated cells. Autoradiograms, such as those in panel A, were scanned and the areas of the 1.6 kb bands were determined. Each point represents the mean (± 1 S.D.) of three independent experiments.



Repair of Individual Strands of the LTL Insert

Even though no differences are observed in repair of both strands (together) of LTL, following hormone induction, differences may be observed in repair of individual strands of this insert, as observed for all other transcribed genes examined in rodent cells (see Hanawalt and Mellon, 1993). Therefore, we investigated strand-specific repair of LTL, again using the method of Bohr et al.((5) , and complementary probes to individual strands of the tk gene. As shown in Fig. 7A, repair of CPDs in untreated cells was almost complete in each strand of LTL following 24 h of repair incubation after 12 J/m^2 UV light. Quantitation of these autoradiograms, expressed as the percent of CPDs removed, is shown in Fig. 8(top panel) along with the time course for recovery of tk mRNA (dotted line). The data indicate that there is little difference in the extent of repair of CPDs after 24 h in either strand with or without induction of tk transcription, although a slight increase in repair rate may occur in the TS with dexamethasone treatment (compare open and closed circles in Fig. 8, top panel). Furthermore, the time course for repair is similar to that of the tk mRNA recovery, indicating that transcription-blocking CPDs are included in the sites efficiently repaired in the LTL insert.


Figure 7: Removal of CPDs from individual strands of LTL (A), c-abl (B), and immunoglobulin J chain (C) genes in mouse L1.4-3 cells. A, data are for cells irradiated with 12 J/m^2 UV light. Only the 13.5 kb band is shown for both the template strand (TS) and the coding strand (CS). B and C, data are for cells irradiated with 20 J/m^2 UV light. Only the bands of interest are shown for both the TS and CS of each gene. For each set, Southern blots from 0.6 or 0.9% alkaline agarose gels were hybridized with strand-specific probes, and unirradiated loading standard (1.6 kb) was added to each sample prior to digestion with T4 endo V. The -UV lanes are for samples treated with (+) or without(-) T4 endo V.




Figure 8: Repair of CPDs for individual strands of the integrated LTL construct, and active and inactive genomic genes in mouse L1.4-3 cells irradiated with 12 J/m^2 UV light. In each case, cells were treated with (closed symbols) or without (open symbols) 0.1 µM dexamethasone. Top panel, repair of CPDs in each strand of the HindIII fragment of LTL. After electrophoresis and transfer (as in Fig. 7), membranes were hybridized with DNA probes to the template (circle, bullet) or coding (Delta, ) strands of the tk gene (see Fig. 1). The dotted line is for the return of tk mRNA (taken from Fig. 6B). Bottom panel, repair of the constitutively expressed c-abl gene and the inactive immunoglobulin J chain gene. Data are for the transcribed strand of the BamHI fragment of c-abl (circle, bullet) and both strands of the HindIII fragment of the J chain gene (Delta, ). The data shown are for one set of experiments.



Repair of Active and Inactive Native Genes

It is possible that mouse L1.4-3 cells repair all genomic sequences efficiently, similar to D. melanogaster embryonic cells (de Cock et al., 1991, 1992a, 1992b) and D. discoideum cells (Mauldin et al., 1994). Therefore, we examined whether preferential repair occurs in native genomic sequences in mouse L1.4-3 cells. Repair of CPDs was measured in the constitutively expressed c-abl proto-oncogene (Fig. 1B) and compared to repair in the inactive immunoglobulin J chain gene (Cann et al., 1982). These studies also served as controls for the effect of dexamethasone on the general repair response in these cells as the transcription of these genes is not affected by dexamethasone treatment (Fig. 1B; and Murad, 1991). The rate of repair of the c-abl gene in mouse 3T3 cells has been previously measured by Madhani et al.(1986). Although strand-specific repair was not measured in that study, these authors found that about 60% of the CPDs are removed from actively growing cells, and 85% of CPDs are removed from confluent cells, within 24 h. Fig. 7B shows autoradiograms for the repair of each strand of these two genes, after 20 J/m^2 UV light with and without dexamethasone treatment. Quantitation of autoradiograms such as these, indicates that the c-abl gene is repaired in a strand-specific manner in L1.4-3 cells, and repair of the transcribed strand is similar to that reported for actively growing 3T3 cells (Fig. 8, bottom panel). The nontranscribed strand showed little (or no) repair during the 24-h incubation (Fig. 7B), similar to both strands of the inactive J chain gene (Fig. 8B), as expected for nontranscribed DNA in rodent cells. Finally, dexamethasone treatment had little effect on the repair in each of these sequences (Fig. 8) indicating that, at 0.1 µM concentration, dexamethasone does not markedly effect the normal repair response in these cells.


DISCUSSION

UV Damage in the Integrated LTL Domain

Our results on the distribution of UV-induced CPDs in the inserted LTL construct indicate that the majority of these lesions are localized in the LTR region of intact mouse cells. Combination of in vivo and in vitro dose response curves, as well as sequence analysis for potential thymidine dimer (TD) sites, indicates that CPD formation may occur at a higher frequency in the MMTV-LTR than reported previously for other defined sequences (e.g. see Evans et al., 1993; Venema et al., 1990a). The extent of damage to the tk gene seen by Northern blot analysis (>85% of transcription blocked after 20 J/m^2 UV) in a transcript that is only 1.6 kb in size also suggests there are ``hotspots'' for UV damage in the LTL domain. In addition, the fact that this inhibition follows single-hit theory (Fig. 4) suggests that the inhibition of transcription is a direct result of UV damage to LTL DNA.

Both the primary sequence of LTR (e.g. long pyrimidine tracts) and the conformation of DNA in chromatin may contribute to this inhibition in vivo. Formation of CPDs has been shown to be modulated by (a) flanking sequences (e.g. Gordon and Haseltine, 1982; Mitchell, et al. 1992), (b) DNA conformation (e.g. Becker and Wang, 1989; Tang et al., 1991), (c) nucleosome formation (Gale et al., 1987; Gale and Smerdon, 1988), and (d) binding of transcription factors (Pfeifer et al., 1992). The phased array of six nucleosomes in the MMTV-LTR may ``fix'' the path of the DNA helix around the histone octamer (Piña et al., 1990a), yielding favorable stacking of pyrimidine bases at specific sites for formation of CPDs or other UV photoproducts (Gale and Smerdon, 1988; Pfeifer et al., 1992).

Although there is good evidence that CPDs in the path of RNA polymerase block transcription (Selby and Sancar, 1990), little is known about the effect of UV damage in regulatory regions on formation of the initiation complex. In the case of the LTL construct, blockage of transcription may occur at the initiation of transcription, since most of the transcribed region is GC-rich and there is a clear bias for CPD formation in the LTR promotor (Fig. 3). In the receptor-binding domain of LTR, a nucleosome precisely positions two of the four GREs, which are accessible to the hormone-receptor complex (Beato et al., 1991). Furthermore, KMnO(4) interference studies indicate that receptor binding is very sensitive to modification of two thymidines in the TGTTCT motifs within the GRE (Truss et al., 1990). Thus, CPD formation in this region may alter hormone-receptor binding, which is required for the induction of transcription. Experiments are currently underway to test this hypothesis.

DNA Repair in the Integrated LTL Domain

This study was undertaken with the expectation of finding enhanced repair of CPDs in LTL in the transcriptionally induced state and a lower rate of repair in the uninduced state (see Introduction). However, even when transcription is induced greater than 50-fold from a very low constitutive level, there is no increase observed in the overall rate of repair of the LTL sequence, and equivalent rates of repair are observed for both the transcribed and nontranscribed strands (Fig. 8). (^2)The efficient repair of both strands of LTL is not due to background replicative synthesis, since isolation of unreplicated DNA (by the density-shift technique) and inhibition of replicative synthesis (with hydroxyurea) during repair yield similar results as untreated cells (e.g.Fig. 5B). Furthermore, the removal of CPDs occurs rapidly in the transcribed strand of c-abl, much more than in the nontranscribed strand (Fig. 7B) or either strand of the inactive J chain gene (Fig. 8). Therefore, transcription-coupled repair occurs in these cells. Furthermore, the nontranscribed strand of c-abl and both strands of the J chain gene are inefficiently repaired in the presence or absence of hormone ( Fig. 7and Fig. 8). Thus, although we observe very efficient repair in both strands of the LTL insert (not coupled to transcription), transcription-repair coupling exists in other genes in these cells, in agreement with numerous studies on pol II genes (e.g. see Hanawalt and Mellon, 1993; Friedberg et al., 1994).

Northern blots showed no (measurable) transcripts from the nontranscribed strand of the tk gene (data not shown). This was the case for probes to either the tk or LTR sequences. Furthermore, a dsDNA probe to the entire LTL yielded only the 1.6- and 3.8-kb transcripts of the tk gene. Therefore, it is unlikely that efficient repair of the nontranscribed strand of the tk gene results from transcription of sequences in that strand of LTL (although it is possible that regions very close to the LTL insert are transcribed).

The lack of transcription-repair coupling in LTL most likely reflects either (a) the strong bias of CPD formation in the LTR promotor or (b) some unique feature(s) of the LTR chromatin. If the 4-fold bias (discussed earlier) of CPD formation in the LTR promotor occurs in intact cells, a 6:1 ratio of CPDs in LTR over tk gene would be expected (for the three LTR and two tk sequences in LTL). Thus, repair of CPDs in LTL may reflect primarily repair of the LTR promotor, and this region may lack transcription-repair coupling while the tk gene does not. Alternatively, the LTL insert may have unique features in the mouse chromatin, which ``influence'' the efficiency of repair enzymes. These features could reflect an unusual primary structure of the viral sequences in LTL (e.g. no introns), and/or the chromatin position in which the LTL has inserted. Indeed, the rate of DNase I digestion of LTL chromatin in the absence of dexamethasone is greater than for the J chain gene (Zaret and Yamamoto, 1984), indicating that LTL DNA may be in a ``poised'' chromatin configuration even in the absence of tk transcription. Thus, the LTL construct may have inserted into a very efficiently repaired region, and its repair is modulated by adjacent chromatin structure (analogous to position effect variegation).

Finally, we have shown that the rate of DNA repair at specific CPD sites in a yeast minichromosome varies markedly (Smerdon and Thoma, 1990). Five different transcripts are made from this minichromosome, and four of the five templates show a good correlation between rate of transcription and rate of repair (Bedoyan et al., 1992; Smerdon et al., 1993). The fifth template region, however, is transcribed very weakly and, yet, is repaired very rapidly (Bedoyan et al., 1992; Smerdon et al., 1993). This region of the minichromosome was found to contain two very unstable nucleosomes in water (i.e. the conditions used for repair experiments; Bedoyan et al., 1992). Thus, the specific chromatin structure of the MMTV-LTR may also affect the coupling of repair and transcription in the LTL construct.


FOOTNOTES

*
This study was supported by National Institutes of Health Grant ES04106 from the National Institute of Environmental Health Sciences. 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.

§
Present address: Biology and Chemistry Dept., Battelle Pacific NW Laboratory, P7-56, Box 999, Richland, WA 99352.

Present address: Dr. Daniel Den Hoed Kliniek, Medical Oncology Dept., P. O. Box 5201, 3008 AE Rotterdam, The Netherlands.

**
To whom correspondence should be addressed. Tel.: 509-335-6853; Fax: 509-335-9688.

(^1)
The abbreviations used are: CPD, cis-syn cyclobutane pyrimidine dimer; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; HSV, herpes simplex virus; tk, thymidine kinase; LTL, LTR-tk-LTR fusion construct; GRE, glucocorticoid response element; T4 endo V, T4 UV endonuclease V; TS, transcribed strand; CS, coding strand.

(^2)
Lower values for the extent of repair of LTL were initially calculated for these cells (Murad, 1991; Smerdon et al., 1993); however, further analysis at lower UV doses and repeat analysis of previous data indicate a higher level of repair occurs, while confirming the lack of transcription-coupled repair.


ACKNOWLEDGEMENTS

We thank Drs. Kenneth Zaret and Keith Yamamoto for providing the L1.4-3 cell line and pLTL1 plasmid used in this study, Dr. Jean Wang for providing plasmid pBS-Ty4, and Dr. Marian Koshland for providing plasmid pJc21. We also thank Drs. Kristoffer Valerie and Jon de Riel for providing the pTACdenV plasmid used for isolation of T4 endo V, and Dr. R. Stephen Lloyd for providing purified T4 endo V. Finally, we thank Drs. Antonio Conconi and Raymond Reeves for critically evaluating this manuscript.


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