(Received for publication, September 1, 1994; and in revised form, November 16, 1994)
From the
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.
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) ()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
-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.
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).
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.
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 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 (
,
) and HindIII (
,
) 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 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
(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
) 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 (
) and tk gene (
)
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
10
and 1.6
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.
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 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
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).
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 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
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 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
. 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 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.
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 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
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 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 (
,
) or coding (
,
) 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 (
,
) and both strands of the HindIII fragment
of the J chain gene (
,
). The data shown are for one set of
experiments.
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 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.
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.