Mammalian DNA mismatch repair protects cells from UVB-induced DNA damage by facilitating apoptosis and p53 activation

DNA Repair 2 (2003) 427–435

Mammalian DNA mismatch repair protects cells from UVB-induced DNA damage by facilitating apoptosis and p53 activation Anthea C. Peters a , Leah C. Young a , Tomoko Maeda b , Victor A. Tron b,c , Susan E. Andrew a,c,∗ a

Department of Medical Genetics, University of Alberta, 8-33 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7 b Department of Laboratory Medicine and Pathology, University of Alberta, 4B1.19 MacKenzie Health Sciences Centre, Edmonton, Alberta, Canada T6G 2R7 c Department of Experimental Oncology, University of Alberta, Alberta, Canada T6G 2H7 Accepted 13 December 2002

Abstract DNA mismatch repair (MMR) is integral to the maintenance of genomic stability and more recently has been demonstrated to affect apoptosis and cell cycle arrest in response to a variety of adducts induced by exogenous agents. Comparing Msh2-null and wildtype mouse embryonic fibroblasts (MEFs), both primary and transformed, we show that Msh2 deficiency results in increased survival post-UVB, and that UVB-induced apoptosis is significantly reduced in Msh2-deficient cells. Furthermore, p53 phosphorylation at serine 15 is delayed or diminished in Msh2-deficient cells, suggesting that Msh2 may act upstream of p53 in a post-UVB apoptosis or growth arrest response pathway. Taken together, these data suggest that MMR heterodimers containing Msh2 may function as a sensor of UVB-induced DNA damage and influence the initiation of UVB-induced apoptosis, thus implicating MMR in protecting against UV-induced tumorigenesis. © 2003 Elsevier Science B.V. All rights reserved. Keywords: MMR; Apoptosis; UVB; p53 activation; Mouse embryonic fibroblasts; Msh2

1. Introduction The mammalian DNA mismatch repair (MMR) system repairs mispaired bases and insertion/deletion Abbreviations: MMR, mismatch repair; MEF, mouse embryonic fibroblasts; O6 -meG, O6 -methyl guanine; NER, nucleotide excision repair; UVB, ultraviolet (290–320 nm); Tag, T antigen; PARP, poly (ADP-ribose) polymerase-1; FACS, fluorescence activated cell sorter; PI, propidium iodide; TCR, transcription coupled repair ∗ Corresponding author. Tel.: +1-780-492-1127; fax: +1-780-492-1998. E-mail address: [email protected] (S.E. Andrew).

loops acquired during DNA replication. Mammalian MMR heterodimers MutS␣ (Msh2 and Msh6) or MutS␤ (Msh2 and Msh3) recognize and bind to such DNA damage, and MutL heterodimers recruit additional proteins required for repair of the adducts. Loss of one of these proteins, is sufficient to confer a mutator phenotype and subsequent cancer predisposition to both humans and mice, underscoring the importance of MMR in maintaining genomic stability (reviewed in [1]). In addition to repairing post-replicative errors, MMR proteins bind some DNA adducts formed by exogenous substances. For some exogenous agents

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MMR is required to mediate removal of the adduct, while for others MMR does not influence repair, but is required for appropriate apoptosis and cell cycle arrest. For example, although MMR binds to both CDDP-induced DNA damage and O6 -methylguanine (O6 -meG), loss of MMR confers complete resistance to the cytotoxic effects of O6 -meG and partial resistance to CDDP [2–6]. Such partial or complete resistance in MMR-deficient cells often corresponds with a failure to apoptose in response to DNA damage [6–10]. Also, MMR has also been shown to facilitate cell cycle arrest in the G2 -M transition in response to DNA damage [11–13]. Therefore, depending on the type of DNA damage, loss of MMR may result in increased mutagenesis, loss of cell cycle control, and resistance to apoptosis, all of which promote neoplastic transformation. Cumulative evidence suggest that MMR may be involved in protective cellular responses to UV-induced DNA damage, but likely does not mediate removal of these adducts in mammalian cells. MutS␣ binds to UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) in vitro, but has not been show to influence removal of these adducts [5,14]. Both CPDs and 6-4PPs are etiological factors for non-melanoma skin cancer (NMSC) [15] and are repaired primarily by nucleotide excision repair (NER). The data addressing the role of MMR proteins in transcription coupled repair (TCR) of CDPs is complicated by opposing results. In eukaryotic systems, such as S. cerevisiae [16,17], mouse [18,19], and human [5] cells that are not tumor-derived, MMR has not been demonstrated to influence the TCR of CPDs. Conversely, repair assays in tumor-derived cell lines have shown MMR-dependant TCR of CPDs [9,20], although there is evidence to the contrary [21]. However, the tumors used to generate these cell lines certainly acquired genetic mutations other than loss of MMR and, as cell lines tend to be used at high passage numbers, the MMR-deficient cell lines could be assumed to have acquired more mutations in vitro than the MMR-proficient controls due to their inherent mutator phenotype. Moreover, although MMR does not appear to be required for repair of UVB-induced adducts, Msh2-null mice are predisposed to UVB-induced skin tumorigenesis compared to wild-type controls [22]. Thus compelling evidence does suggest that MMR may be involved in cellular

responses to UV-induced DNA damage other than repair, and its ability to recognize photoproducts as well as its role in tumor suppression points to a role for MMR in sensing UV damage and signaling the apoptotic response. Inactivation of the tumor suppressor p53 has been shown to be an early event in the development of NMSC [23], and mice deficient in p53 are predisposed to UV-induced squamous cell carcinomas [24]. Activation of p53 occurs in response to UVB-induced DNA damage by phosphorylation of serines, such as 15, 20 and 392, which may be important in post-UV cell cycle arrest, apoptosis and NER [25–27]. However, UVB-induced apoptosis may occur through both p53-dependent and -independent pathways [1]. Similarly, MMR mediates apoptosis through both p53-dependent [28,29] and delayed p53-independent mechanisms [7] in response to O6 MeG adducts; p53 phosphorylation in response to O6 MeG adducts is MMR-dependent [30]. In order to determine if MMR plays a role in mediating the UVB-induced apoptotic response in mammalian cells, we compared the post-UVB responses of Msh2-null and wildtype mouse embryonic fibroblasts (MEFs). We found, in both primary and SV40-transformed MEFs, that Msh2-null MEFs were partially resistant to the cytotoxic effects of UVB, and that this resistance was coupled with a reduction in apoptosis. Furthermore, the absence of Msh2 also resulted in a reduction in the phosphorylation of p53, a possible transducer of the apoptotic signal. These data show that Msh2 may function as a damage sensor of photoproducts or contribute to the processing of UVB-induced DNA damage, thereby, initiating apoptosis and influencing the activation of p53. 2. Materials and methods 2.1. Msh2 mouse colony Mice deficient in Msh2, previously generated by gene targeting were genotyped as previously described [31] with the following modifications. The PCR reaction included the following reagents: 1× PCR buffer without Mg (Invitrogen), 15 mM MgCl2 , 1.25 mM dNTPs, 5 ␮l primer L926 (50 pmol), 3 ␮l primer U771 (30 pmol), 3 ␮l primer neo (30 pmol),

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2.5 U Taq polymerase (Invitrogen) and 3 ␮l of 3/100 dilution of tissue digestion to a final volume of 50 ␮l. Primers U771 (5 GCT CAC TTA GAC GCC ATT GT 3 ) and L926 (5 AAA GTG CAC GTC ATT TGG A 3 ) amplify a 174 bp fragment of the wildtype allele, and primers U771 and neo (5 TGG AAG GAT TGG AGC TAC GG 3 ) amplify a 300 bp fragment identifying the neo gene of the target vector. The PCR thermocycle conditions were as follows: 95 ◦ C for 2 min; 40 cycles of 95 ◦ C for 30 s, 58 ◦ C for 1 min, 72 ◦ C for 30 s. PCR products were separated by agarose gel electrophoresis (2% (w/v), TAE buffer) containing ethidium bromide for visualization of PCR product. 2.2. Cell culture Primary mouse embryonic fibroblasts (MEFs) were cultured with Dulbucco’s minimal essential medium (DMEM) plus 20% fetal bovine serum (FBS) (Gibco BRL). Day 13 embryos were dissociated in trypsin. The primary cells were split 3:1 prior to reaching confluence and only low passage number cells (<4 passages total) were for experimentation. SV40 Large T antigen (Tag)-transformed MEFs BC1-6 (Msh2+/+ ) and MS5-7 (Msh2−/− ) [32] were cultured with DMEM plus 10% FBS, and cells were used for experiments no more than four passages from original frozen stock. 2.3. UVB irradiation Media was aspirated from 80% confluent cell cultures (unless otherwise noted), cells were rinsed with PBS and exposed to the UVB (290–320 nm) source, a bank of six unfiltered UVB bulbs (FS20T12/UVB-BP, Light Sources Inc., Orange CT), and culture media was replaced. Control cells were subjected to a mock UV treatment. The intensity of the UVB source penetrating the culture dish lid was determined by placing a culture dish lid over the sensor of an IL1700 radiometer with a SED 240/UVB-1/W detector (International Light, Newburyport, MA). Culture dish lids filtered out contaminating UVC radiation [33]. 2.4. Assay for overall cell survival Primary MEFs at 80% confluence in 35 mm plates were treated with UVB. Forty-eight hours post-UVB,

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the plates were washed twice with PBS and cells were stained with crystal violet in 20% methanol. Stained plates were scanned and subjected to densitometry (Quantity One, Bio-Rad). 2.5. Colony formation assay BC1-6 and MS5-7 cells were plated at 250 cells per 60 mm culture dish. Plates were irradiated within 6–12 h, incubated for 7 days, and stained with crystal violet. Colonies with >50 cells were scored, and expressed relative to non-treated controls. The assay was repeated a minimum of three times (with triplicates for each dose) and standard error of the mean was determined. 2.6. Apoptosis Forty-eight hours post-UVB, adherent and suspension cells were collected. The percentage of apoptotic cells was determined by FACs analysis using an Annexin-FITC Apoptosis Kit (BD Pharmingen, San Diego, CA). Cells were analyzed for cleavage of poly(ADP-ribose) polymerase-1 (PARP) and caspase 3 by western blot as described later. 2.7. Western blot analysis Cell pellets were sonicated in lysis buffer (50 mM Tris pH 7.5, 10 mM MgCl2 , 1× protease inhibitor (Complete Mini, Roche), 1% SDS). Protein concentrations were determined using a detergent compatible protein assay (BioRadDC). Cell lysates were separated by discontinuous SDS-PAGE and electro-transferred to PVDF membrane in Tris/glycine buffer. Membranes were blocked in 5% non-fat milk in TBST at room temperature for 1 h. Primary antibodies, incubated in 5% milk/TBST, were as follows: Msh2 (clone FE11/Ab-2, Oncogene Research Products, Boston, MA), Msh6/GTBP (clone 44, BD Transduction Laboratories), cleaved caspase 3 (Asp175) (Cell Signaling Technology, Beverly, MA), PARP (clone C2-10, PharMingen International), p53 (Ab-3, Oncogene Research Products), phospho-p53 ser15 (#9284, Cell Signaling Technology, Beverly, MA), p42 MAP Kinase 3A7/Erk2 (Cell Signaling Technology, Beverly, MA). Antibody was visualized using the appropriate HRP-conjugated secondary antibody and the Western

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Lightning luminol reagent (Perkin Elmer, Boston, MA) on Fuji SuperRX film (Fujifilm, Stamford, CT).

3. Results 3.1. Msh2-null MEFs show partial resistance to UVB-induced cytotoxicity The cytotoxic effects of UVB were investigated using both an overall cell survival assay for the primary MEFs and a colony formation assay for the SV40-transformed MEFs. In both cases Msh2-null MEFs exhibited a significantly (P < 0.05) higher level of survival post-UVB as compared to their isogenic wildtype controls (Fig. 1, n ≥ 3). 3.2. Msh2-null cells undergo less post-UVB apoptosis than their wild-type counterparts To investigate further the role of MMR in post-UVB survival, UVB induced apoptosis was measured by staining irradiated MEFs with labeled AnnexinV and PI. Analysis of the stained MEFs by FACS (Fig. 2) revealed for both the primary and transformed MEFs, that the Msh2-null MEFS underwent significantly reduced levels of apoptosis (Annexin positive, PI negative) post-UVB (P < 0.05 and 0.01, respectively). Fig. 2a depicts apoptosis 24 h post-UVB in the primary MEFs; Fig. 2b depicts apoptosis 24 h post-UVB in the transformed MEFs. Comparing Fig. 2a (primary MEFs, 24 h) and Fig. 2b (transformed MEFs, 24 h), it can be seen that the transformed MEFs undergo higher levels of apoptosis than the primary cells at 100 J/m2 . Reduction in UVB-induced apoptosis in Msh2-null primary and transformed MEFs was confirmed by western blot analysis of the cleavage products of caspase 3 and PARP (Fig. 3). In the both the transformed and primary MEFs there was more caspase 3 cleavage products (17 and 20 kDa) in the wildtype than in the Msh2-null MEFs. The antibody used for the detection of cleaved caspase 3 does not recognize full length caspase 3. Similarly, there was more cleavage of PARP in the wildtype MEFs that in the Msh2-null MEFs.

Fig. 1. Msh2-null primary and transformed MEFs exhibit increased survival post-UVB. (A) Assay for overall survival: Msh2-null and wildtype primary MEFs were irradiated with 0 to 250 J/m2 of UVB and 48 h post-UVB the plates were stained and analyzed for density to indicate relative overall survival of the MEFs. (B) Colony formation assay: Transformed MEFs irradiated with 0–50 J/m2 UVB were scored for colonies with >50 cells. Data represents at least two or three, independent experiments, respectively, of 3 plates per dose. Data is expressed as the mean of the data ±S.E.M. Comparisons were made using a paired Student’s t-test. ∗ P < 0.05, ∗∗ P < 0.01.

3.3. Phosphorylation of p53 is reduced or delayed in Msh2-null MEFs To determine the influence of Msh2 on p53 stabilization and activation post-UVB, we investigated its level of expression and the amount of p53 phosphorylated on murine serine 18 (corresponding to serine 15 in humans). After exposure to 100 J/m2 UVB, the levels of p53 phosphorylated at serine 15 were lower in the transformed Msh2-null MEFs that in the

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Fig. 2. Msh2-deficiency confers partial resistance to UVB-induced apoptosis. (A) Percent early apoptosis (Annexin V+ /PI− ) of wild-type and Msh2-deficient primary MEFs 24 h post-UVB. (B) Percent early apoptosis (Annexin V+ /PI− ) of transformed MEFs BC1-6 (Msh2+/+ ) and MS5-7 (Msh2−/− ) 24 h post-UVB. Data is expressed as the mean of the data ±S.E.M. (n ≥ 3). Comparisons were made using a paired Student’s t-test. ∗ P < 0.05, ∗∗ P < 0.01.

control MEFs (Fig. 4). This result was also observed at 200 J/m2 but the difference was less apparent (data not shown). Moreover, the total levels of p53 were also lower in transformed Msh2-null MEFs (MS5-7) than in the wildtype control MEFs (BC1-6) (Fig. 4a). In the primary MEFs, there was a pronounced and reproducible difference in the phosphorylation of p53 8 h post-UVB, but by 24 h this difference was no longer evident (Fig. 4b). In contrast to the transformed MEFs, the overall levels of p53 were equivalent between the Msh2-null and wildtype primary MEFs post-UVB. 4. Discussion An increasingly broad spectrum of DNA adducts have been shown to activate a MMR-dependent path-

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Fig. 3. Msh2-null MEFs express reduced levels of caspase 3 and PARP cleavage. Primary and transformed MEFs were treated with 200 J/m2 UVB and subjected to Western blot analysis. Data are representative of at least two independent experiments. Caspase 3 cleavage during apoptosis generates 20 and 17 kDa fragment (full length caspase 3 not detected by this antibody) and PARP (116 kDa) cleavage generates an 85 kDa fragment. (A) Caspase 3 cleavage in wildtype (+) or Msh2-null (−) primary MEFs. (B) Caspase 3 cleavage in transformed wildtpe MEFs (BC1-6, denoted by +) and Msh2-null MEFs (MS5-7, denoted by −). (C) PARP cleavage in transformed wildtype MEFs (BC1-6, denoted by +) and Msh2-null MEFs (MS5-7, denoted by −).

way of apoptosis [1], thus it is reasonable to hypothesize that its repertoire extends to UV-induced DNA adducts. Previous studies have implicated MMR in cellular responses to UV-induced DNA damage, however no direct link between MMR and UV-induced apoptosis has yet been shown. The present study provides evidence that Msh2-deficient MEFs show increased survival and a reduced capacity for apoptosis in response to UVB, thus supporting a role for MMR in mediating the apoptotic response to UV damage. Our data also implicates Msh2 in facilitating p53 activation by phosphorylation on serine 15 in response

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Fig. 4. p53 expression and activation in primary and transformed MEFs post-UVB. Whole cell lysates from UVB treated (100 J/m2 ) transformed (A) and primary (B) MEFs were subjected to Western blot analysis and analyzed for overall levels of p53 and levels of p53 phosphorylated on serine 15.

to UVB, further supporting the importance of MMR in the post-UVB cellular response. Mammalian MMR does not appear to play a direct role in TCR of UV-induced DNA damage [5,18,19], despite controversy from earlier studies in cancer cell lines reporting the contrary [9,20]. Cells deficient in TCR of UV-induced DNA adducts exhibit a phenotype of marked sensitivity to UVB [34]. In contrast, we have shown that Msh2-null cells exhibit tolerance to UVB-induced cytotoxicity (Fig. 1), indirectly supporting the argument that Msh2 is not required for TCR. Furthermore, we investigated the effect of Msh2-deficiency in global genomic repair of UVB-induced DNA adducts and were unable to show a consistent difference in repair between either Msh2−/− or Msh6−/− MEFs and their isogenic wildtype controls.1 Thus the effects of MMR-deficiency in cellular responses to UVB radiation are not likely attributable to defective DNA repair. Both the transformed and primary Msh2-null MEFs exhibited a significant increase in clonogenic survival post-UVB (P < 0.05) (Fig. 1b). In previous experi1

Peters, unpublished data and Young, unpublished data.

ment using these transformed MEFs it was observed that loss of Msh2 conferred high-level resistance to the alkylating agent MNU and low-level resistance to IR [32]. Our data indicate that loss of Msh2 confers moderate resistance to UVB irradiation. Previously reported sensitivity to UVC does not seem to be a reproducible characteristic of MMR-deficient mammalian cells [3,9,20,35]. Several studies have shown differences in biological responses to UVC versus UVB, including differential gene transactivation by p53 [36] and a lower ratio of CPDs to oxidized purines [37], both of which may affect the role of MMR in cell survival. Our report is the first to investigate the clonogenic response of MMR-deficient cells to UVB radiation, the UV range most responsible for the sun’s carcinogenic effects [15]. The absence of Msh2 conferred a significant reduction in the apoptotic response post-UVB in primary and transformed MEFs, supporting a role for Msh2 in the UVB DNA damage response (Figs. 2 and 3). The observation that MMR deficiency did not completely abolish apoptosis indicates that Msh2 does not play an exclusive role in initiating apoptosis. Although DNA damage is an undisputed stimulant of UVB-induced apoptosis, UV does affect other molecular targets that are also capable of inducing apoptosis independent of the nuclear triggers, such as the death receptor of the tumor necrosis factor receptor superfamily CD95 (Fas, APO-1) [38]. It is possible that deficiency of Msh2 in our transformed and primary MEFs abrogated the nuclear stimulus for UV-induced apoptosis, but that an independent basal level of death receptor-induced apoptosis occurs equally in both Msh2-proficient and -deficient cells, thus accounting for the remaining apoptosis observed in the Msh2-null MEFs. Alternatively, MMR may be integral to the response of normal cells to the secondary oxidative damage caused by UV-irradiation, which is an important contributor to the mutational effects of UV irradiation. The observation that MMR is important to the regulation of apoptosis in response to treatment with hydrogen peroxide [8], is consistent with this model. We also observed that at moderate UVB doses (for example, 100 J/m2 in Fig. 2) apoptosis levels were higher in the transformed MEFs than in the primary MEFs. Tag has been hypothesised to inhibit apoptosis through its ability to debilitate both the transactivation

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ability of p53 and p300 (a co-activator of p53), as well as though its homology to the anti-apoptotic protein Bcl-2 [39]. A recent study by Cole et al. [39] found that Tag-induced sensitization of rat embryo fibroblasts (REF) to 5-FU was dependent on the integrity of the J domain which facilitates binding of Rb [40]; these cells also remained capable of p53-dependent apoptosis despite being bound by Tag. Therefore, the involvement of p53 in MMR-mediated post-UVB apoptosis in our transformed MEFs is uncertain, but experiments are underway in cells with genetic deficiencies in both MMR and p53 to clarify these interactions. Our results, however, do implicate the involvement of p53 downstream in MMR-mediated post-UVB. We found that serine 15 phosphorylation is reduced or delayed in the absence of Msh2 in both primary and transformed MEFs post-UVB (Fig. 4). Duckett et al. [30] found that phosphorylation of p53 at serine 15 and 392 in response to O6 MeG requires MMR, although the mechanism for this interaction has not been determined. They also reported that UVC-induced phosphorylation of serine 392 of p53 was independent of hMSH6 status, but did not investigate the role of hMSH2 or hMLH1 in this process [30]. Our finding suggests that in response to UVB, Msh2 influences phosphorylation on serine 15, a modification critical for the p53 stress-response [24]. AT-related kinases ATM or ATR are responsible for phosphorylation of p53 on serine 15 in response to mainly IR and UV, respectively [41]. ATM, and possibly ATR, have been found to exist in a large protein complex along with hMSH2 and hMSH6 [42]. Therefore, a possible signaling pathway for MMR-mediated post-UVB apoptosis may involve activation of ATR followed by activation of p53. Also, induction of the p53-related gene p73 has been show to require functional both MMR and c-abl after cisplatin exposure [6] and may be important to the post-UVB response. Recently it has been suggested that the binding of MMR proteins to a DNA lesion may be important required for the recruitment of ATM and CHK2, thus bringing them into proximity of each other and allowing for activation of CHK2 by ATM [43]. Alternatively, MMR may indirectly contribute to apoptosis post-UVB through an involvement in the processing of UVB-induced lesions, thus, giving rise to secondary lesions that trigger apoptosis [44].

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Although it is known that recognition of several types of DNA damage by MMR proteins result in a signal transduction cascade that contributes to levels of apoptosis and/or cell cycle arrest, the mechanisms which influence this cascade have not been identified [29]. Overexpression of MSH2 or MLH1 has been demonstrated to induced apoptosis [45]. Also, MSH6 and, to a lesser extent MSH2, have been found to be phosphorylated by protein kinase C (PKC) [46], which is required for normal response of the cells examined to UV [47]. In summary, we have shown that Msh2-null MEFs have a reduced ability to undergo apoptosis and increased survival following UVB exposure. Although the resistance to UVB-induced apoptosis conferred by MMR-deficiency may not be as dramatic as the resistance conferred to apoptosis induced by alkylating agents [32], Msh2-null mice are more susceptible to UVB induced skin cancer than wildtype control mice [48], suggesting that the level of MMR-dependent apoptosis post-UVB reported here are physiologically relevant. Additionally, we found that Msh2-null MEFs have a delayed or diminished activation of p53. Although this reduced activation of p53 could contribute to the lower levels of apoptosis observed in the Msh2-null MEFs, additional research in our laboratory has indicated that Msh6-null MEFs also demonstrate reduced apoptosis, but do not exhibit altered activation.2 Studies to elucidate the extent of p53-dependent and p53-independent apoptotic mechanisms that are regulated by MMR are underway. These data support our hypothesis that MMR contributes to the apoptotoic response of cells to UVB radiation and that Msh2 may play an important role in preventing UV-induced skin cancer without necessarily being directly involved in the repair of UV-induced DNA adducts.

Acknowledgements We wish to thank Le Luong, Eugene Chomey, Melanie Boskill, and Gabi Constantinescu for their technical assistance. This work was funded by the National Cancer Institute of Canada (NCIC). AP is 2 Young et al., submitted for publication and Young, unpublished data.

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supported by the Natural Sciences and Engineering Research Council (NSERC) and the Alberta Heritage Foundation for Medical Research (AHFMR). L.C. Young is supported by the Alberta Cancer Board (ACB), and SEA is a Canadian Institutes of Health Research (CIHR), Canadian Genetic Diseases Network (CGDN), and AHFMR scholar.

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