Department of Biology, McMaster University, Hamilton,
1280 Main Street West, Ontario, Canada L8S 4K1
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Abstract |
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Abbreviations: Ad, adenovirus; -MEM,
-minimal essential medium; ß-gal, ß-galactosidase; CS, Cockayne syndrome; HCMV, human cytomegalovirus; HCR, host cell reactivation; JNK, c-jun N-terminal kinase; NER, nucleotide excision repair; RNAPII, RNA polymerase II; TCR, transcription-coupled repair; BER, base excision repair; XP, xeroderma pigmentosum.
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Introduction |
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Two complementation groups of CS (CS-A and -B) have been identified, each of which exhibits a defect in TCR, while the bulk repair sub-pathway of NER appears to function normally (1). XP is composed of a minimum of seven complementation groups (XP-AG), each displaying a general deficiency in NER which compromises at least the bulk DNA repair sub-pathway and usually TCR as well. The exception is XP-C, which has been shown to retain viable TCR in spite of severely compromised bulk repair (2,3). Although CS and XP are clinically distinct disorders, some individuals from each of three XP groups (B, D and G) exhibit symptoms characteristic of CS, defining a clinical XP/CS complex (47).
The XPB and XPD proteins have each been shown to be components of transcription factor TFIIH (8,9) which plays a role in both NER and transcription by RNA polymerase II (RNAPII) (10). The CSA and CSB proteins co-immunoprecipitate (11) and CSA has been shown to interact with TFIIH directly (11) while CSB can do so via XPG (12). Although these interactions and the dual role of TFIIH have led to the suggestion that the clinical presentation of CS may arise primarily from a defect in transcription rather than in repair (13,14), recent evidence has indicated a specific repair defect in these cells (15). TFIIH, XPG and the RPA/XPA/XPF/ERCC1 complex (16,17) are required for both sub-pathways of NER, whereas the XPC/HHR23B complex (18) only appears to be required for the repair of bulk DNA (2,3,19).
CS cells are also deficient in the repair of ionizing radiation induced damage in the transcribed strand of an active gene (20). Products of ionizing radiation such as thymine glycols are removed by the base excision repair (BER) pathway (reviewed in 21) and are removed in a transcription-associated manner in normal cells (22), suggesting a role for the CS gene products in coupling BER to transcription. TCR of oxidative damage, which is repaired by the BER pathway, also requires the function of the XPG protein which is distinct from its endonuclease activity that is associated with NER (23).
Examination of cellular repair generally requires that the cells are damaged in some manner, which makes it difficult to determine if the repair pathways are constitutively active or induced by the damaging agent. However, pretreatment of a variety of mammalian cells with various physical and chemical DNA damaging agents results in an increased survival (or enhanced reactivation) for several nuclear replicating viruses damaged by UV or ionizing radiation. It has been suggested that enhanced reactivation of DNA-damaged viruses results from an induced DNA repair pathway (24,25). Additionally, UV-enhanced reactivation of UV-damaged simian virus 40 (SV40) in CV-1 African green monkey cells has been shown to result from an enhanced restoration of early viral gene function (26) suggesting that UV irradiation of the cell enhances repair and/or allows circumvention of DNA lesions in early viral genes. More recently, we and others have shown that host cell reactivation (HCR) of a UV-damaged reporter gene is inducible by UV (2729), as well as heat shock (30) in human fibroblasts and implicated a role for wild-type p53 in this process (28,29).
Other evidence for damage-induced DNA repair pathways in mammalian cells comes from a number of studies including the enhanced DNA repair capacity of mammalian cells following carcinogen treatment (31), the p53-mediated enhancement of excision repair by the DNA damage induced GADD45 gene (32,33) and the identification of a novel DNA repair response which is induced by irradiation of cells at the G1/S border (34). Mammalian cells also exhibit a hyper-sensitivity to X-rays at low doses but increased radio-resistance at higher doses (35,36). The increased radio-resistance is absent in some DNA repair-deficient lines (35), and the hyper-sensitivity can be removed by pretreatment of cells with `priming' doses of X-rays or hydrogen peroxide (36), suggesting an inducible DNA repair response in mammalian cells. A recent report indicates that exposure of human cells to `priming' doses of ionizing radiation 4 h prior to a higher dose leads to the enhanced removal of thymine glycols after the higher dose (37), providing evidence for an inducible BER response in human cells.
The recombinant adenovirus vector Ad5HCMVsp1lacZ contains the ß-galactosidase (ß-gal) reporter gene under the control of the human cytomegalovirus (HCMV) immediate early promoter, inserted into the deleted E1 region of the virus (38). This vector can efficiently infect and express the reporter gene in primary human cell strains, but is unable to replicate in the absence of exogenous E1 expression. A preliminary report of work with this construct indicated that HCR of the UV-damaged reporter is severely deficient in untreated primary XP-C cells (27), even though this complementation group has previously been shown to efficiently repair the transcribed strand of active genes (3). However, following UV irradiation of the host cells prior to infection, HCR of the UV-damaged reporter was enhanced to near normal levels in XP-C fibroblasts (27).
We have also demonstrated (39) that although XP-C fibroblasts exhibit a normal capacity to support adenovirus (Ad) DNA synthesis following UV exposures of >9 J/m2 to the cells, these strains display a reduced capacity for Ad DNA synthesis following lower UV exposures. Conversely, CS strains exhibit normal capacity for Ad DNA synthesis following low UV exposures, but are significantly reduced in this capacity following exposures of >9 J/m2. Ad DNA replication requires host gene products (40) and this assay is believed to measure the ability of the host cells to remove transcription-blocking lesions from critical cellular genes in order to permit viral replication (39). These observations are consistent with a UV-inducible mechanism for the repair of the transcribed strand of active genes and suggest that enhanced reactivation of a reporter construct is representative of cellular processes. Here, we further examine UV-enhanced HCR of the UV-damaged reporter construct in three XP-C fibroblast strains, as well as in strains from XP groups B, D, F and G, and CS groups A and B.
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Materials and methods |
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Ad5HCMVsp1lacZ is a non-replicating Ad5 derived virus expressing the lacZ gene under control of the HCMV immediate early promoter. This construct expresses ß-gal in various human cell types without replication of the virus (38). Viral stocks were prepared as described previously (41).
UV irradiation of virus
UV irradiation of the virus has been described (24). Viral suspensions in 1.8 ml of unsupplemented -MEM were irradiated in 35 mm dishes on ice with continuous stirring using a General Electric germicidal lamp (model G8T5) emitting predominantly at 254 nm at an incident fluence rate of 2 W/m2 (J-255 shortwave UV meter, Ultraviolet Products, San Gabriel, CA). Aliquots of 200 µl were removed for each exposure to the virus and diluted appropriately with unsupplemented medium.
UV enhanced reactivation of the reporter gene
Cells were seeded at a density of 1.9x104 cells/well in 96-well microtitre plates (Falcon, Lincoln Park, NJ). Between 20 and 24 h after seeding, media was aspirated from microtitre plates and replaced with 40 µl warmed phosphate-buffered saline [140 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4 and 1.75 mM KH2PO4 (pH 7.4)] per well. Cells were then UV irradiated (or mock irradiated) using a General Electric germicidal lamp (model G8T5) emitting predominantly at 254 nm at an incident fluence rate of 1 W/m2. UV exposures employed were corrected for irradiation in 96-well dishes as previously reported (39). Immediately following UV irradiation, cells were infected with either UV-irradiated or non-irradiated Ad5HCMVsp1lacZ in a volume of 40 µl at a multiplicity of infection of 10 plaque forming units/cell. Following viral adsorption for 90 min at 37°C, the cells were re-fed with warmed supplemented medium.
Infected cells were harvested at 4044 h after infection. Infected cell layers were incubated 20 min at 37°C in 250 mM Tris, 1 µM PMSF, 0.5% NP-40 (pH 7.8), followed by 10 min in 100 mM sodium phosphate, 10 mM KCl, 1 mM MgSO4, 50 mM 2-mercaptoethanol (pH 7.5) (32). A405 was determined at several times following addition of O-nitrophenol ß-D-galactopyranoside (0.1% ONPG) using a 96-well plate reader (Labsystems multiscan MCC/340 and/or Bio-Tek Instruments EL340 Bio Kinetics Reader).
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Results |
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HCR of ß-gal activity for UV-irradiated Ad5HCMVsp1lacZ is enhanced by UV pre-treatment in normal and XP-C cell strains, but not in strains from other XP or CS complementation groups
HCR of ß-gal activity for UV-irradiated Ad5HCMVsp1lacZ was also examined in UV-irradiated fibroblasts. Typical survival curves for HCR of ß-gal activity for UV-irradiated Ad5HCMVsp1lacZ in fibroblasts from each of the complementation groups following representative UV exposures and no UV exposure to the cells are presented in Figure 2. It can be seen that prior UV irradiation of a normal cell strain and an XP-C strain resulted in a significant enhancement of HCR of ß-gal expression from the UV-damaged reporter gene. In contrast, HCR of the UV-damaged reporter gene in other XP groups was inhibited by prior UV irradiation of the infected cells and HCR of the UV-damaged reporter in the CS strains appeared to be unaffected by prior UV irradiation of the cell over the range of UV fluences examined (Figure 2
). D37 values for the UV survival of ß-gal activity of UV-irradiated Ad5HCMVsp1lacZ were calculated and used to quantitate the relative HCR of the UV-damaged reporter gene following each UV exposure to cells. The results from multiple experiments have been combined and the average relative HCR values for each cell strain (± SEM) are presented in Figures 3 and 4
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Discussion |
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Basal HCR (i.e. in non-irradiated cells) of the UV-damaged reporter gene for most of the repair deficient cell strains examined was significantly reduced compared with that in normal strains (Figure 1; Table I
). This included at least two XP-C cell strains, a complementation group characterized previously as proficient in the repair of actively transcribed genes. Both UV irradiation (27,28; this paper) and heat shock (28,30) of normal and XP-C cell strains resulted in enhanced HCR of the UV-damaged reporter gene. However, prior UV irradiation of cells from the other XP and CS complementation groups examined did not result in enhanced HCR of the UV-damaged reporter (Figures 2 and 4
). Similarly, heat shock enhanced HCR of the UV-damaged reporter was not detected in XP-A, XP-D, XP-G or CS strains (30). As the products of the XP-A, XP-B, XP-D, XP-F, XP-G, CS-A and CS-B, but not the XP-C gene are all required for the preferential repair of active genes, the lack of UV or heat shock enhanced HCR of the UV-damaged reporter in these strains suggests that efficient TCR is necessary for this process.
It is possible that the treatment of cells with UV or heat shock induces an increased processivity of RNAPII allowing it to bypass or read through some UV-induced DNA lesions. This would result in an increased reactivation of a UV-damaged reporter gene in treated normal cells relative to their non-treated counterparts. However, we consider this explanation very unlikely, since an inducible transcriptional read-through mechanism cannot account for the impaired ability of non-irradiated XP-C cells to reactivate the UV-damaged reported gene that we have observed (27,30; this work). The most likely explanation of our data is that the observed UV-enhanced reactivation of the UV-damaged reporter gene results from an induced DNA repair process on the damaged reporter gene.
Previous work examining gene specific repair in NER deficient fibroblasts generally used UV exposures to the cell on the order of 10 J/m2 to induce measurable numbers of adducts in specific cellular gene fragments (13,19,4850). Relative HCR values for the various NER deficient strains for cells given prior UV exposure of 10 J/m2 are comparable with these previously reported measurements of TCR. However, our results suggest that the levels of gene-specific repair observed following these UV exposures to the cells might be different from those in cells which have received no UV or lower UV exposures.
Non-irradiated fibroblasts from XP complementation groups B and F appear to retain considerable ability to reactivate the UV-damaged reporter, although this activity rapidly disappears upon prior irradiation of the cells. Prior UV irradiation of the cell is also seen to result in a small reduction in HCR of the UV-damaged reporter gene in XP-D and XP-G cells. Cellular UV irradiation induces expression from the HCMV immediate early promoter, and we have observed that ß-gal expression from the undamaged reporter construct is enhanced following UV exposure of both TCR-proficient and TCR-deficient cell strains (51). This suggests that the decreases in HCR of the UV-damaged reporter observed in TCR-deficient cell strains following cellular UV irradiation are not due to a loss of expression of transcription factors driving the ß-gal gene. It is possible that following UV irradiation the relatively low residual repair capacity of these cells for the UV-damaged reporter gene is competed away by damage in the cellular genome. Alternatively, the reduction in repair of the UV-damaged reporter gene may arise from a decrease in the number of viable cells following UV exposure of these strains during the course of the experiment.
XP-C cells have been reported to be proficient in NER of UV-induced DNA damage for the transcribed strand of active genes, although deficient in the repair of both the non-transcribed strand of active genes and inactive or bulk DNA (3). Therefore, we were initially surprised to find a deficiency in basal HCR of the UV-damaged reporter gene in several XP-C cell strains. Similarly surprising were the relatively high levels of basal HCR observed in the CS strains (5790% of normal levels), as these cells have been shown to be deficient in the repair of the transcribed strand of active genes following UV irradiation of the cell (50). HCR of the UV-damaged reporter gene was significantly enhanced by prior UV irradiation of all normal and XP-C cell strains. In contrast, no enhancement in HCR of the UV-damaged reporter gene was observed in the CS strains following UV irradiation of the cells. These results suggest that in the absence of sufficient UV exposure to the cell, damage in the reporter gene is repaired in normal cells to a large extent by the bulk DNA sub-pathway of NER, although some TCR must also occur as evidenced by the sub-normal HCR values in non-irradiated CS cell strains. However, following sufficient UV stimulation of the cells it appears that TCR or a process dependent on TCR is activated, resulting in an increased repair of the transcribed strand of the reporter gene in XP-C and normal cells.
A large degree of heterogeneity was observed in the HCR values for non-irradiated XP-C fibroblasts. Heterogeneity in repair phenotype has been noted previously for this complementation group. Negligible preferential repair was detected in the XP-C cell strain XP4RO (48), while near normal levels for the repair of the DHFR and ADA genes were noted in the XP-C strain XP1TE (3), and intermediate levels of gene specific repair were observed in the XP-C cell strains XP1BE and XP2BE (19). It is possible that these differences result from different levels of activation of the TCR pathway in different cell strains. Additionally, as experimental conditions such as UV (2729; this paper), heat shock (28,30) and pH (M.A.Francis and A.J.Rainbow, unpublished observations) can all affect the level of repair of a UV-damaged reporter construct it is also possible that other conditions such as oxidative stress, or even degree of cell confluence may affect the UV exposures required to induce TCR or the levels of TCR observed.
Even after the UV enhancement of HCR in the XP-C strains, the HCR levels observed did not attain the HCR levels observed in normal cell strains. Two of the XP-C cell strains used in this work (XP1BE and XP2BE) have previously been shown to have somewhat less efficient repair on the transcribed strand of the DHFR gene compared with that in normal strains following UV exposure, although still much more efficient than repair of the non-transcribed strand, or an inactive locus (19). This small deficiency in repair of the transcribed strand in UV-irradiated XP-C cells, as well as the inability of the XP-C strains reported in this paper to attain normal levels of HCR for the UV-damaged reporter gene following prior UV irradiation, may result from a significant level of repair of active genes by the bulk repair pathway in normal cells under these conditions.
Although CS has been characterized as a deficiency in the preferential repair of active genes, the CS-A strain CS3BE removes the lesions induced in the ADA and DHFR genes by 10 J/m2 with ~50% of the efficiency of normal cells (1), and a similar repair ratio was also observed for a CS-B strain (49). Likewise, for prior UV exposure of 10 J/m2 to cells, the HCR values for the UV-damaged reporter gene in three of the four CS strains examined were ~50% of the normal controls (Figure 4). The one exception, GM2965, was observed to exhibit uncharacteristically high HCR levels. However, this individual has been characterized as an atypical CS patient due to late age of onset of symptoms and the lack of ocular and retinal lesions (52), consistent with a milder repair defect in this individual. Taken together these data suggest that in the absence of an inducing stimulus, the bulk repair sub-pathway of NER accounts for 5080% of the repair in active genes. However, upon stimulation of the cells, TCR is induced resulting in the increased levels of repair in active genes.
HCR of a UV-damaged reporter gene is believed to reflect the ability of cells to repair an actively transcribed gene. However, even though XP-C cells retain preferential repair of active genes and CS cells do not, the levels observed for HCR of the UV-damaged reporter construct are similar for several XP-C and CS strains following 1015 J/m2 UV exposure to the cells. However, these observations are not necessarily contradictory. Levels of preferential repair are determined by comparing repair in an active gene to that of an untranscribed region. Thus, even though XP-C and CS cells may exhibit similar levels of repair in the transcribed strand of an active gene, a non-preferential repair phenotype would be assigned to CS because this repair level is the same as that of inactive DNA. In contrast, because XP-C cells exhibit compromised bulk DNA repair, even sub-normal levels of gene-specific repair would still present a preferential repair phenotype.
Squires and Johnson (53) have quantified levels of incision (an early step of NER) following UV irradiation of human fibroblasts and demonstrated that in cells from normal, XP-D and XP-G individuals strand breaks accumulate immediately following radiation. In contrast, the accumulation of incisions was seen to be delayed 15 min following irradiation of XP-C fibroblasts. This result is consistent with a UV-inducible excision pathway, and suggests that the signal is mediated within 15 min which indicates that intracellular signaling pathways, rather than activation of transcription, accounts for this induction. We have previously shown that wild-type p53 is required for heat shock and UV-enhanced HCR of the UV-damaged reporter construct (28). The p53 protein has been shown to interact with the XP-B, XP-D, and CS-B proteins (54), to modulate the activity of TFIIH (54), and to be phosphorylated in response to UV by a kinase characteristic of a c-jun kinase (JNK) (55). JNK, in turn, has been shown to be activated by both UV-induced DNA lesions (56) and heat shock (57), suggesting a possible role for this kinase in the UV and heat shock induced reactivation of a UV-damaged reporter gene.
In addition to the UV enhanced reactivation of a UV-damaged reporter gene under the control of the HCMV promoter reported by us and others (2729), thymine dinucleotides have been shown to induce the reactivation of a UV-damaged reporter gene under the control of the SV40 early promoter, by a process which may also involve p53 (58). More recent results by Huang et al. suggest that transcription from a p53 driven promoter in the presence of wild-type p53 results in upregulation of both transcription and repair of a UV-damaged reporter gene, and that the enhanced DNA repair of the reporter gene is a separate, distinct activity of p53, but is dependent on p53 driven transcription (59). Since UV exposure to human fibroblasts results in the accumulation and increased activity of p53 (44,45,60) and there may be as many as 200300 p53 consensus binding sites in the human genome (61), there could be a large number of cellular genes that are preferentially repaired in response to UV damage through a concomitant upregulation of transcription and repair by a p53-dependent mechanism.
Whereas Huang et al. report that p53 expression had no such effect on a reporter gene driven by promoters lacking a p53 recognition element, such as the SV40 early promoter, others have shown that repair of a UV-damaged reporter gene driven by the SV40 promoter is increased in the presence of wild-type p53 (32,62,63). We and others have shown that repair of the transcribed strand of a UV-damaged reporter gene under the control of the HCMV immediate early promoter is inducible by UV (2729), as well as heat shock (30) in human fibroblasts, and that the induced repair is dependent on wild-type p53 (28,29). Since the HCMV promoter used in our reporter gene construct (64) and the SV40 promoter used in the reporter gene construct of others (32,62,63) contain no p53 consensus binding sites, it appears that there may be more than one mechanism whereby UV exposure to cells can trigger an enhanced repair in the transcribed strand of certain active genes. The mechanisms by which p53 upregulates nucleotide excision repair on the transcribed strand of some expressed genes in response to UV and other cellular stresses remains to be determined. While these mechanisms need to be investigated further, such mechanisms would give a priority for DNA repair to a subset of actively transcribed genes (possibly including genes essential for cell survival) following exposure to UV and other cellular stresses.
In summary, the evidence presented in this paper suggests that, not only does the UV-inducible repair of a UV-damaged reporter gene involve the TCR sub-pathway of NER, but also, that in the absence of sufficient UV exposure to cells (or other appropriate stimulus) to induce this pathway, the damage in the reporter gene is repaired to a large extent by the bulk DNA repair sub-pathway of NER.
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Notes |
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Acknowledgments |
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References |
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