Carcinogenesis vol.20 no.8 pp.1389–1396, 1999
COMMENTARY
Potential roles for p53 in nucleotide excision repair
Bruce C.McKay2, Mats Ljungman and Andrew J.Rainbow1 Department of Radiation Oncology, Division of Cancer Biology, University of Michigan Comprehensive Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0936, USA and 1Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 2To whom correspondence should be addressed Email:
[email protected]
Ultraviolet (UV) light-induced DNA damage is repaired by the nucleotide excision repair pathway, which can be subdivided into transcription-coupled repair (TCR) and global genome repair (GGR). Treatment of cells with a priming dose of UV light appears to stimulate both GGR and TCR, suggesting that these processes are inducible. GGR appears to be disrupted in p53-deficient fibroblasts, whereas the effect of p53 disruption on TCR remains somewhat controversial. Normal recovery of mRNA synthesis following UV irradiation is thought to depend on TCR. We have found that the recovery of mRNA synthesis following exposure to UV light is severely attenuated in p53-deficient human fibroblasts. Therefore, p53 disruption may lead to a defect in TCR or a postrepair process required for the recovery of mRNA synthesis. Several different functions of p53 have been proposed which could contribute to these cellular processes. We suggest that p53 could participate in GGR and the recovery of mRNA synthesis following UV exposure through the regulation of steady-state levels of one or more p53regulated gene products important for these processes. Furthermore, we suggest that the role of p53 in the recovery of mRNA synthesis is important for resistance to UV-induced apoptosis. Introduction DNA is constantly exposed to damaging agents from either endogenous or exogenous sources. Several highly conserved DNA repair pathways have evolved to reduce the consequences of exposure to this damage. Ultraviolet light (UV) induces primarily cyclobutane pyrimidine dimers (CPD) and pyrimidine(6–4)pyrimidone dimers and these lesions are substrates for the nucleotide excision repair (NER) pathway. The rate of repair of UV dimers is heterogeneous throughout the genome, with repair occurring more rapidly in the transcribed strands of active genes than in the remainder of the genome. This repair process is termed transcription-coupled repair (TCR) and it is thought that TCR permits the rapid resumption of RNA synthesis following UV irradiation (1). It is well established that fibroblasts derived from patients Abbreviations: CPD, cyclobutane pyrimidine dimers; CS, Cockayne syndrome; GGR, global genome repair; HCR, host cell reactivation; HPV-E6, human papilloma virus 16 E6; LFS, Li–Fraumeni syndrome; NER, nucleotide excision repair; RRS, recovery of mRNA synthesis; TCR, transcriptioncoupled repair; UV, ultraviolet; XP, xeroderma pigmentosum. © Oxford University Press
with xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are defective in their capacity to repair UV-induced DNA damage. Of particular relevance to the present commentary, XP group C fibroblasts (XP-C) retain the ability to repair by TCR but these cells do not remove UV lesions from the remainder of the genome (1,2). In contrast, cells derived from CS patients in complementation groups A and B (CS-A and CS-B cells) are unable to perform TCR but these cells repair UV lesions by global genome repair (GGR) as efficiently as normal fibroblasts (1,3). Other XP strains, such as XP-A, XP-B, XP-D and XP-G, are unable to repair UV dimers through either pathway (1,2). Efficient TCR in XP-C fibroblasts permits the rapid recovery of RNA synthesis, whereas transcription does not efficiently recover in CS cells or in TCR-deficient XP strains following UV treatment (4–6). In fact, the ability of XP-C but not CS cells to efficiently recover RNA synthesis led to the suggestion that active genes could be repaired more rapidly than the remainder of the genome several years before this was directly demonstrated (7). In the present commentary, evidence will be discussed which suggests that both GGR and TCR may be stimulated by a priming exposure to UV light. Furthermore, the role of p53 in GGR, TCR and the recovery of mRNA synthesis will be discussed. Lastly, we propose that the regulation of basal levels of one or more proteins by p53 is likely to be important for these cellular processes. Inducible repair of UV photoproducts Recombinant proteins involved in NER have been used to reconstitute NER in vitro (8). However, the efficiency of these in vitro systems to perform NER is low and in vitro excision of UV dimers in chromatin-like substrates is even further attenuated (9). In addition, no in vitro system exists that supports TCR, despite the ability of cell extracts to simultaneously support transcription and repair (10,11). Therefore, not all aspects of TCR or GGR have been faithfully reproduced in vitro, illustrating the complexity of these processes in cells. These pathways may be inducible, which is to say that the full repair potential of cells requires activation of one or more signaling pathways resulting from exposure of cells to stress, including UV light. Several lines of evidence suggest that GGR is inducible. It has been reported that treatment of normal but not XP-A or XP-C cells with a priming dose of UV light enhanced the recovery of semi-conservative DNA synthesis following subsequent UV irradiation (12). The requirement for the XPA and XPC proteins indicates that this enhanced recovery phenomenon is dependent on GGR. Secondly, cell extracts from UV-treated cells support NER better than extracts from unirradiated cells in vitro (13). Also, treatment of cells with thymine dinucleotides prior to UV irradiation increased GGR as assessed by unscheduled DNA synthesis (14). TCR may also be inducible, but without an in vitro model for TCR this question is more difficult to address. However, results obtained 1389
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Fig. 1. UV light and heat shock treatment stimulate host cell reactivation of reporter gene activity. HCR involves the introduction of UV-irradiated lacZ-expressing adenovirus reporter constructs into host cells. After 48 h, β-galactosidase activity is assessed. We have found that HCR requires NER (left). Furthermore, HCR was found to be enhanced by treatment of TCR-proficient but not TCR-deficient cells with either UV light or heat shock prior to infection with UV-irradiated reporter constructs (right) (16,17,20). Therefore, HCR in pretreated cells not only depends on NER but also the induction of TCR and/or a related function such as the recovery of mRNA synthesis (RRS).
with host cell reactivation (HCR) assays suggest that TCR may be inducible (15–19). Enhanced HCR of reporter gene activity Recombinant non-replicating adenovirus constructs have been used to introduce UV-damaged reporter genes into unirradiated primary fibroblasts to assess the repair of UV-induced DNA damage in the absence of cellular stress using HCR of reporter gene activity as an end-point (15–20). UV lesions in the template strand of active genes inhibit progression of RNA polymerase II (21) and a single dimer in a reporter gene is thought to be sufficient to inhibit reporter gene expression (22). Thus HCR of reporter gene activity is thought to require the repair of transcription blocking UV lesions and reflect the TCR capacity of cells. We have shown that HCR of reporter gene activity is reduced in several untreated fibroblast strains, including XP-C (Figure 1; 15,16,19). This is surprising, since XP-C cells are TCR-proficient and would be expected to rapidly remove transcription blocking UV lesions and thus reactivate the UV-damaged reporter gene. Blockage of RNA polymerase II by UV dimers in the reporter construct does not appear to be sufficient to promote the preferential repair of these transcription blocking lesions in XP-C cells (15,16,19). We have shown that treatment of fibroblasts with either UV (5–20 J/m2) or heat shock (43°C for 30 min) stimulated HCR of reporter gene activity in XP-C and normal fibroblasts but not in CS or other XP strains (15,16,19). These results show that HCR is inducible in response to cellular stress and that this induction depends on functional TCR but not GGR. Models of TCR in which stalled RNA polymerase II directly facilitates NER cannot fully account for enhanced recovery of reporter gene activity. Based on these results, we have proposed that efficient TCR requires the activation of one or more stress pathways (16,18,19,23). This model predicts that TCR is inefficient at low UV doses, at which stress pathways would not be expected to be activated. 1390
Dose-dependent variation in TCR TCR is usually assessed by estimating the rate of removal of UV photoproducts from the transcribed strand of a specific gene using lesion-specific endonucleases. This method was initially used to demonstrate gene- and strand-specific repair of CPD from active genes in human cells (24,25) and was instrumental in identifying the specific repair defects in XP-C and CS cells (3,26). This approach requires that a sufficient number of dimers is induced per restriction fragment. TCR has been assessed in 20 and 7 kb DNA fragments following 10 and 30 J/m2, respectively (27). To assess TCR following 1 J/m2, repair would have to be assessed in a 200 kb DNA fragment in order to obtain a sufficient number of lesions per restriction fragment. For this reason, TCR has not been directly assessed at low UV doses. The distribution of repair sites in fibroblast strains with well-characterized DNA repair phenotypes (normal, XP-C, CS-A and CS-B) provides valuable information regarding dose-dependent variation in these NER pathways. It has been recognized for many years that there is a non-random distribution of repair sites following UV treatment in normal and XP-C but not CS fibroblasts (28–33). Whereas DNA repair patches appear to be clustered in XP-C cells at all doses of UV light (28,29,31,34), the ability to detect clustered repair in normal fibroblasts is highly dose dependent (Table I). Results obtained at either low (ø3 J/m2) or high (ù25 J/m2) UV doses suggest that repair is random in the genome of normal fibroblasts (30–32). In contrast, clustered repair is detectable in normal fibroblasts at moderate UV doses typically used to assess TCR (5–20 J/m2) (30–34). As shown in Table I, the ability to detect TCR and clustered DNA repair tightly correlate for all doses of UV light and fibroblast strains examined. Since clustered DNA repair is related to the TCR capacity of cells, it appears that TCR varies in a dosedependent manner. Importantly, the random distribution of
Roles for p53 in nucleotide excision repair
Table I. Correlation between clustered repair and TCR Dose , 5 J/m2
Cell
Normal XP-C CS-A, CS-B XP-A aTCR,
5 ø dose , 25 J/m2
Dose ù 25 J/m2
TCRa
CRb
TCR
CR
TCR
CR
ND ND ND ND
2 1/2c 2 2
1 1 2 2
1 1 2 2
2 1 ND ND
2 1 2 2
the ability to detect a strand preference for repair in an active gene in the indicated cell type and at the indicated dose of UV light (1,3,26,27). to the indicated UV dose in the indicated cells (28–34). was close to random (32).
bCR, the ability to detect clustered repair following exposure cVery little repair detected and the distribution of repair sites
repair sites in normal fibroblasts following low UV doses (30) suggests that TCR is less efficient than GGR at these doses. These results are fully compatible with the concept of inducible TCR. The efficiency with which cells recover mRNA synthesis following UV irradiation is a critical determinant of resistance to apoptosis (6,35). For this reason, one might expect that it would be advantageous for cells to have efficient TCR even at low doses of UV light. However, the results discussed above suggest that TCR is inefficient at low UV doses. There are many potential explanations for this apparent paradox. For example, there are many highly conserved natural transcription pause sites in the genome which are important in the regulation of gene expression. Regulation of transcription in several critical cellular genes is regulated through a prolonged DNA sequence-dependent pause in RNA polymerase II progression (36,37). Thus, the transcription pause sites in these genes are positioned such that they could potentially be substrates for TCR (1,38). As errant cycles of excision and resynthesis may be error prone (39), prolonged pausing might be expected to induce mutations in critical regulatory sequences. Therefore, if the maintenance of conserved pause sites is important for gene regulation, evolutionary pressure may have selected against cells having constitutively active TCR. Cell signaling and TCR As recovery of reporter gene activity from a UV-damaged reporter gene appears to be inducible in response to either heat shock or UV treatment, signaling pathways stimulated by these agents could be important for DNA repair. The role of HSP70 in the repair of UV-induced DNA damage has not been directly assessed in human cells, however, heat shock proteins affect NER in bacteria (40). In support of a heat shock-inducible DNA repair response are the findings that both moderate heat shock and HSP70 expression induce resistance to subsequent UV light exposure in murine keratinocytes, murine fibroblasts, mouse skin and human keratinocytes (41–45). At least a portion of this effect results from a decrease in the extent of UVB-induced apoptosis in mouse skin (45). TCR is protective against apoptosis in human cells in vitro (6,35) and mouse skin in vivo (46). Moderate heat shock has been suggested to stimulate TCR of a UVdamaged reporter gene in primary human fibroblasts (16). Therefore, it is quite possible that the resistance to UV irradiation conferred by either a moderate heat shock or HSP70 expression may result in part from the stimulation of TCR (16). Additional support for some aspect of cell signaling in TCR comes from the assessment of DNA repair synthesis in streptolysin O-permeabilized primary fibroblasts (47). Mild membrane permeabilization disrupted DNA repair synthesis
in normal, XP-C and CS fibroblasts, suggesting that one or more membrane-associated or cytosolic proteins is required for the repair of UV-induced DNA damage, including TCR (47). Also, recent reports suggest that UV light rapidly promotes the redistribution of DNA repair proteins such as XPG, RPA, PCNA and TFIIH and this relocalization may depend, in part, on TCR (48–51). Taken together, these results suggest that TCR is not a passive process but probably requires activation of one or more signaling pathways. p53 and NER The p53 tumor suppressor gene is the most commonly altered gene in cancer (52) and germline transmission of a single mutant p53 allele is frequently associated with Li–Fraumeni syndrome (LFS), a disorder characterized by a predisposition to a variety of cancers (53). The antineoplastic effect of p53 is conferred, at least in part, by inhibiting propagation of cells with unrepaired DNA damage by enhancing DNA repair, promoting cell cycle arrest and/or facilitating apoptosis (54). p53 accumulates in a dose-dependent manner in cells following exposure to genotoxic agents, including UV light, through post-transcriptional mechanisms. The induction of p53 is thought to lead to the transactivation of p53-responsive genes. In addition, p53 may have transactivation-independent functions (54,55). Over the past few years it has become clear that p53 and/ or p53-regulated gene products contribute to the repair of UV-induced DNA damage in both human and mouse cells (13,18,23,56–71). Whereas it is generally accepted that p53 contributes to GGR (59,60,72), the involvement of p53 in TCR is more controversial. Ford and Hanawalt have reported that GGR, but not TCR, is disrupted in two LFS strains and two primary fibroblast strains expressing human papilloma virus 16 E6 (HPV-E6) (58–60). However, studies suggesting that several LFS fibroblast strains are reduced in their capacity to repair UV-induced DNA damage by the TCR pathway are discussed below (18,23,57,64,71). Evidence for the involvement of p53 in TCR Gene-specific (but not strand-specific) removal of T4 endonuclease V-sensitive sites (CPD) from the DHFR and c-myc genes has been assessed in several primary LFS cells which differ from those used by Ford and Hanawalt (57–59,71). The extent of repair observed at 4, 6 and 8 h (particularly at 4 and 6 h) following UV irradiation was ,50% of that observed in control cells, suggesting that removal of CPD from each DNA strand was reduced in these LFS strains compared with control strains. Since repair of CPD appears to have been reduced in both DNA strands, these results imply that TCR of CPD is impaired in these LFS fibroblasts. 1391
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Further evidence for a role of p53 in TCR comes from studies using inhibitors of DNA polymerase δ/ε, such as aphidicolin and 1-β-D-arabinofuranosylcytosine, which block the resynthesis stage of NER. Repair-induced DNA strand breaks generated following post-UV incubation with these inhibitors can be used to quantify excision of UV-induced DNA damage within specific genes in a manner similar to the more commonly used endonuclease-sensitive site assay. Excision of UV-induced DNA damage was found to be reduced in LFS cells both in the genome as a whole and in the c-myc gene, suggesting that LFS cells are deficient in their capacity to excise UV-induced DNA damage by both the GGR and TCR pathways (64). As discussed above, UV- and heat shock-enhanced reactivation of UV-damaged reporter gene constructs appears to reflect the capacity of cells to repair UV-induced DNA damage by TCR (15,16,19). In these studies, UV- and heat shock-enhanced reactivation of reporter gene activity was not detected in LFS cells (18). Similar results were recently reported in UVBtreated murine fibroblasts (69). As reporter gene expression is thought to require the removal of transcription blocking lesions (22), these results support a role for p53 in TCR and the recovery of transcription from a UV-damaged gene. It was recently reported that the ability of UV-irradiated XP-C, but not XP-A, CS-A or CS-B, fibroblasts to support adenovirus DNA replication is similar to the capacity of similarly treated normal fibroblasts (23). We have shown that both primary and immortalized LFS fibroblasts have reduced capacity to support adenovirus DNA synthesis following UV treatment compared with either normal or XP-C fibroblasts (23). Since this biological activity is affected in both p53defective and TCR-defective cells, these results suggest that p53 may play a role in the TCR pathway (23). p53 and the recovery of mRNA synthesis Since proficient TCR is thought to play an important role in the recovery of RNA synthesis following UV irradiation, one would expect that certain LFS- and HPV-E6-expressing cells, which are reportedly proficient in the strand-specific removal of T4 endonuclease-sensitive sites (58–60), would recover RNA synthesis with kinetics similar to that observed in normal fibroblasts. However, we have found that the same LFS and HPV-E6-expressing fibroblasts used by Ford and Hanawalt (59,60) have a severe defect in the recovery of mRNA synthesis following exposure to the same dose of UV light (73). Consistent with our results, it has also been reported that other primary LFS cells are defective in the recovery of total nascent RNA synthesis (64,71) and this is probably related to a defect in the recovery of mRNA synthesis. Furthermore, results obtained using virus-based assays of DNA repair suggest that the capacity of LFS cells to recover mRNA synthesis from UV-damaged DNA is impaired (18,23). Taken together, recent results suggest that LFS cells do not recover mRNA synthesis efficiently following UV irradiation. Although it remains unclear whether p53 or p53-regulated gene products participate in TCR per se, experiments that either directly (73) or indirectly (18,23,64,71) assessed the recovery of mRNA synthesis suggest that TCR, or a postrepair process required to recover mRNA synthesis, is impaired in LFS cells. The loss of endonuclease-sensitive sites has been interpreted to indicate that CPD have been repaired completely, however, this assay reflects the incision and pre-incision steps of NER and does not necessarily indicate that DNA repair 1392
Fig. 2. Potential roles for p53 in NER and RRS. p53 may participate in NER and the RRS through either transactivation-dependent functions or transactivation-independent functions of p53. Transactivation-independent functions of p53 may involve protein–protein interactions between p53 and proteins involved in DNA repair. Whereas transactivation-dependent functions of p53 may be required to regulate the expression of one or more proteins limiting for NER and RRS. p53 could potentially contribute to these processes even in unstressed cells (A). Following UV irradiation (B), p53 accumulates, which may stimulate the direct interaction with DNA repair proteins. However, the transactivation-dependent functions of p53 will be impeded by the presence of transcription blocking lesions in the p53 target genes (B).
is complete. Therefore, it remains unclear whether delayed recovery of mRNA synthesis in p53-deficient cells is the consequence of a defect in TCR or in another process required for the recovery of transcription following UV irradiation. Potential roles for p53 in NER and the recovery of mRNA synthesis There are two general mechanisms through which p53 could contribute to NER and the recovery of mRNA synthesis (Figure 2). First, p53 is a well-characterized activator of transcription of p53-responsive genes (54) and may contribute to the regulation of proteins involved in these cellular processes (Figure 3). Second, p53 may be capable of interacting with proteins or DNA damage (Figure 3B; 54) in a manner which facilitates NER and the recovery of mRNA synthesis. It is important to consider the possibility that p53 may participate in cellular functions in either the absence of cellular stress (Figure 2A) or following cellular stress (Figure 2B). Although p53 is generally considered to be in a latent form in the absence of cellular stress (54), p53 may be able to act as a transcriptional regulator in the ‘uninduced’ as well as the ‘induced’ state (Figure 2; 54,74). Similarly, a direct participation of p53 in DNA repair (such as DNA damage binding or protein–protein interaction) could also involve ‘uninduced’ and/or ‘induced’ p53 (Figure 2). These scenarios will be considered separately below.
Roles for p53 in nucleotide excision repair
Fig. 3. p53 binds DNA repair proteins and transactivates genes encoding DNA repair proteins. (A) p53 binds to several proteins which have been suggested to play a role in DNA repair, including PCNA, RPA, XP-B, XP-D and CS-B. These have been reported to be involved in GGR and TCR, respectively. Other proteins, such as BRCA1 and BRCA2, also interact with p53 and may contribute to DNA repair (92–95). (B) The promoters of several potential TCR and GGR genes have p53 response elements and some of these have been shown to be positively regulated by p53.
Direct participation of p53 in global genome repair? Although it is commonly stated that p53 contributes to GGR, disparate results have been obtained in different model systems. Whereas one strain of p53 null murine fibroblasts are reportedly GGR proficient (75), other p53 null fibroblast and keratinocyte strains have clear GGR defects (67–69). Furthermore, GGR is impaired in immortalized LFS fibroblasts (58) but some p53 null tumor cell lines, such as HL60 and A253 cells, retain significant levels of GGR (76,77). Finally wild-type p53 expression has been shown to complement the GGR defect in immortalized LFS cells (58) whereas expression of p53 in SAOS-2 osteosarcoma cells which lack endogenous p53 does not increase UV-stimulated unscheduled DNA synthesis or the rate of UV-induced excision (78,79). Thus, these seemingly conflicting results question the direct involvement of p53 in GGR. p53 is known to bind directly to several DNA repair proteins required for GGR (XPB, XPD and RPA) and TCR (XPB, XPD, CSB and RPA) (Figure 3A; 13,18,57,69,80). However, these interactions may be more important for the regulation of apoptosis than for the repair of UV-induced DNA damage (81). As indicated above, the relatively efficient repair of UV lesions in several p53 null tumor cells argues against a requirement for p53 in GGR. Thus, although GGR appears to be affected by p53 disruption in certain cells, it is uncertain what (if any) role p53 protein, either at a basal or induced level, plays in the NER process (Figure 2).
UV light-induced expression of p53-regulated proteins Bacterial cells respond to a variety of DNA-damaging agents and other cellular stresses by increasing expression of a variety of gene products through a transcriptional derepression mechanism. This response leads to enhanced DNA repair, enhanced mutagenesis and enhanced survival (82). It is tempting to speculate that p53 enhances repair of UV-induced DNA damage in a manner analogous to the SOS response (83) by increasing the expression of one or more gene products which contribute to NER (Figure 3B). Certain proteins, such as p21waf1, GADD45, p48 and PCNA, are positively regulated by p53 (54,55,84). Furthermore, the promoter of the mismatch and transcription-coupled repair gene MSH2 contains p53 consensus binding motifs and may be regulated by p53 (85). Thus, p53 could indirectly stimulate DNA repair by increasing the expression of one or more limiting proteins involved in NER (Figure 2). Two arguments can be made against a role for the UVinduced transactivation of p53-responsive genes in the enhancement of NER. Firstly, UV-induced DNA damage blocks the elongation of RNA polymerase II and, thus, UV lesions will inhibit expression of p53-responsive genes (Figure 2). In fact, decreased p21waf1 mRNA and protein levels have been observed following UV irradiation in human cells (35,86) and the time required for the recovery and induction of p21waf1 protein levels inversely correlate with the TCR capacity of cells (35). Thus for the UV-induced expression of p53responsive genes to contribute to DNA repair, UV-irradiated cells would require the removal, modification or bypass of transcription blocking lesions to permit expression of these genes (35,87). Importantly, this implies that CS cells would have reduced levels of GGR, since these cells are defective in TCR and the recovery of mRNA synthesis. However, CS cells have no defect in GGR, suggesting that the recovery of mRNA synthesis does not significantly contribute to GGR. Secondly, both GGR and TCR appear to be insensitive to cycloheximide treatment (88,89), whereas TCR but not GGR is sensitive to α-amanitin treatment (89,90). Taken together, these two observations argue against a transactivation-dependent role for p53 in the enhancement of GGR or TCR following UV irradiation. However, this argument does not exclude a possible role for p53 in the regulation of steady-state levels of proteins important for the repair of UV-induced DNA damage and the recovery of mRNA synthesis (Figure 2A). p53 and the steady-state regulation of p53-responsive genes To date, only three reports have demonstrated that an inducible DNA repair or related activity was disrupted in p53-deficient cells (13,18,69). However, these reports fail to discriminate between a requirement for p53 either prior to or following UV irradiation. This is a critical issue because the levels of p53regulated proteins in unstressed cells have been shown to be affected by the basal level of p53 (74). Recently, it was reported that expression of the p48 gene (disrupted in XP group E) is regulated by wild-type p53 even in the absence of DNA damage (83). Importantly, the NER phenotype of p48deficient XP-E fibroblasts appears to be similar to that of LFS cells (71,84). Furthermore, the kinetics of recovery of total nascent RNA synthesis is similar in these cell types (71), suggesting that the regulation of p48 protein levels by p53 may be important for both NER and the recovery of mRNA synthesis. As already discussed, the UV-induced expression of gene products required for NER and the recovery of mRNA 1393
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Fig. 4. Does p53 direct the preferential repair of its target genes? Recently Huang et al. (70) reported that co-transfection of a UV-damaged reporter gene with a p53 expression vector resulted in enhanced reactivation of reporter gene activity. Importantly, p53-enhanced reactivation was only observed if the promoter contained a p53 response element (p53-RE). These results suggest that p53 could facilitate repair of responsive genes by recruiting DNA repair proteins to its target genes. Such a model might be expected to facilitate expression of p53-regulated genes, such as p21waf1, which are important for survival and resistance to UV-induced apoptosis (35,91).
is not fast enough to contribute to these recovery processes at early times following UV irradiation (35). We suggest that p53 stimulates NER and the recovery of mRNA synthesis by regulating the steady-state levels of one or more proteins (such as p21waf1 and/or p48) that are limiting for these recovery processes. Does TCR vary in a gene-specific manner? It was recently reported that co-transfection of a plasmid expressing p53 and a UV-damaged plasmid containing a reporter gene under control of a p53-responsive promoter greatly stimulated HCR of reporter gene activity (70). As already discussed, p53 contributes to NER and the recovery of total mRNA synthesis, therefore, it is likely that at least two functions of p53 contribute to this apparent preferential recovery phenomenon. One function is probably p53stimulated DNA repair. The second function may involve the preferential targeting of NER to p53-responsive genes (Figure 4). We have found that p21waf1 expression is attenuated following high doses of UV light but the recovery of expression and subsequent induction of p21waf1 was only observed in TCR-proficient fibroblasts (35). These results clearly demonstrate the importance of DNA repair to the UV-enhanced expression of p53-regulated gene products (35). Interestingly, significant heterogeneity in the rate of repair of the transcribed strand of different genes has recently been reported (75). This raises the possibility that other transcription factors may be effective in the recruitment of NER proteins for the preferential recovery of expression of target genes. Such a mechanism might be expected to enhance resistance to UV-induced apoptosis by increasing expression of UV-protective gene products such as p21waf1 (35,66,91). Thus, in addition to its role in enhancing apoptosis (54), p53 may confer protection against UV-induced apoptosis under certain conditions. Acknowledgements We would like to thank Todd Bulmer, Murray Francis and Jim Stavropoulos (Department of Biology, McMaster University) for helpful discussions and critical reading of the manuscript. B.C.M. and M.L. would also like to thank members of the Department of Radiation Oncology at the University of Michigan. A.J.R. is supported with funds from the National Cancer Institute of Canada. M.L. is supported by a grant from the University of Michigan Comprehensive Cancer Center’s Institutional Grant from the American Cancer Society.
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