Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts

Hsiang-Tsui Wang; Bongkun Choi; Moon-shong Tang

doi:10.1073/pnas.1005244107

Communicated by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, April 23, 2010 (received for review December 30, 2009)
↵¹H.-T.W. and B.C. contributed equally to this work.

Abstract

Melanomas occur mainly in sunlight-exposed skin. Xeroderma pigmentosum (XP) patients have 1,000-fold higher incidence of melanoma, suggesting that sunlight-induced “bulky” photoproducts are responsible for melanomagenesis. Sunlight induces a high level of reactive oxygen species in melanocytes (MCs); oxidative DNA damage (ODD) may thus also contribute to melanomagenesis, and XP gene products may participate in the repair of ODD. We examined the effects of melanin on UVA (320–400 nm) irradiation-induced ODD and UV photoproducts and the repair capacity in MC and XP cells for ODD and UV-induced photoproducts. Our findings indicate that UVA irradiation induces a significantly higher amount of formamidopyrimidine glycosylase-sensitive ODD in MCs than in normal human skin fibroblasts (NHSFs). In contrast, UVA irradiation induces an insignificant amount of UvrABC-sensitive sites in either of these two types of cells. We also found that, compared to NHSFs, MCs have a reduced repair capacity for ODD and photoproducts; H₂O₂ modified- and UVC-irradiated DNAs induce a higher mutation frequency in MCs than in NHSFs; and, XP complementation group A (XPA), XP complementation group C, and XP complementation group G cells are deficient in ODD repair and ODD induces a higher mutation frequency in XPA cells than in NHSFs. These results suggest that: (i) melanin sensitizes UVA in the induction of ODD but not bulky UV photoproducts; (ii) the high susceptibility to UVA-induced ODD and the reduced DNA repair capacity in MCs contribute to carcinogenesis; and (iii) the reduced repair capacity for ODD contributes to the high melanoma incidence in XP patients.

It has long been recognized that sunlight exposure is the major cause of skin cancers, including melanoma (1–3). Although evidence from both epidemiological and molecular studies show that photoproducts induced by UVB (290–320 nm) irradiation are the major cause for nonmelanoma skin cancer (4), epidemiological studies strongly implicate UVA (320–400 nm) irradiation in sunlight exposure-related cutaneous melanomagenesis (5, 6). In animal models, it has been found that both UVA and UVB irradiation can trigger melanocytic hyperplasia and melanomagenesis (7–9). Although UVB radiation can induce both cyclobutane pyrimidine dimers (CPDs) and pyrimidine < 6–4 > pyrimidone photoproducts (<6–4 > PPs), which can trigger mutagenesis and carcinogenesis (10, 11), the photoproduct yield of these types of DNA damage by UVA irradiation is two to three orders of magnitude lower than by UVB radiation (12). Thus, exactly how UVA acts to promote melanomagenesis remains controversial. It has been found that XP patients have 1,000-fold higher incidence of melanoma than normal individuals (13). Xeroderma pigmentosum cells are sensitive to UV radiation but resistant to ionizing radiation, and XP gene products are also crucial for CPD and <6–4 > PP repair (14). Therefore, it is possible that UVA irradiation of melanocytes (MCs) may induce “bulky” photoproducts that are repairable by a nucleotide excision repair (NER) mechanism and these photoproducts, if not repaired, trigger mutagenesis and, consequently, melanomagenesis.

It is well established that the major effect of UVA irradiation is the induction of reactive oxygen species (ROS) and that melanin can greatly enhance this process (15–17). It has been proposed that UVA-induced oxidative stress (OS) and oxidative DNA damage (ODD) are the two major contributors to UVA-induced melanomagenesis (18). The occurrence of mucosal melanoma in regions that are rarely exposed to sunlight—such as genitalia, the colon, and the nasal septum (19–21)—suggests that DNA damage other than photoproducts is also involved in melanomagenesis. If we hypothesize that ODD is the culprit responsible for triggering mucosal melanomagenesis, then one has to assume MCs in these tissues are undergoing endogenous OS and generating ROS, even without sunlight irradiation. Because melanin is an effective free radical scavenger and would presumably mitigate OS, the role of melanin in melanomagenesis remains unclear (22).

Previously, using host cell reactivation (HCR) and in vitro repair synthesis assays, we have found that OS and lipid peroxidation (LPO) byproducts—such as 4-hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA), and acrolein (Acr) —inhibit NER and enhance mutagenesis through direct modification of repair proteins by the carbonyl group in the aldehydes (23–25). These findings raise the possibility that the intrinsically high OS in melanocytes and OS induced by UVA irradiation may induce LPO; LPO byproducts could then reduce the DNA repair capacity, which in turn could contribute to melanomagenesis.

To understand the role of melanin in UVA radiation-induced DNA damage formation and the mechanism that leads to high melanoma incidence in XP individuals, we determined the effects of melanin on UVA-induced ODD and UV photoproduct formation; the repair capacity for ODD and UV photoproducts in MC, XPA, XPC, and XPG cells; and the mutagenicity of ODD in these cells. Our results show that melanin plays a crucial role in UVA-induced mutagenesis and also, most likely, in melanomagenesis.

Results

Oxidative DNA Damage and Bulky Photoproducts Induced by UVA Irradiation in MCs.

It has been shown that melanin enhances free radical generation, including reactive ROS resulting from UVA irradiation (15–17). Hence, it is possible that melanin enhances ODD. To test this possibility, lightly and darkly pigmented MCs and normal human skin fibroblasts (NHSFs, CRL2097) were irradiated with UVA light and the formation of ODD was detected by the formamidopyrimidine glycosylase (Fpg) incision assay. Formamidopyrimidine glycosylase incises 8-oxo-deoxyguanosine (8-oxo-dG) and the imidazole ring-opened adducts of purines; these adducts are generated in cells under OS and also in H₂O₂- treated DNA (26). The results in Fig. 1A show that UVA irradiation does indeed enhance Fpg-sensitive site formation in both lightly and darkly pigmented MCs, but not in NHSFs, indicating that UVA irradiation induces ODD in MCs but not in NHSFs. We used the UvrABC incision method to determine the effect of melanin on CPD and <6–4 > PP induction by UVA. It is well established that UvrABC, the NER enzyme complex, can specifically and quantitatively incise CPDs and <6–4 > PPs (27, 28). The results in Fig. 1B show that genomic DNAs isolated from UVA-irradiated MCs and NHSFs are only slightly, if at all, sensitive toward UvrABC nuclease, which indicates that melanin does not affect UVA irradiation-induced bulky photoproduct formation. It is worth noting that UvrABC does not recognize H₂O₂-modified DNA and Fpg does not recognize UVC-induced bulky photoproducts (Fig. S1). Based on the results shown in Fig. 1, we concluded that UVA irradiation induces ODD in MCs but not in NHSFs, and that the frequency of UvrABC-sensitive sites formed in UVA-irradiated MCs is much lower than the frequency of Fpg-sensitive sites formed in the same cells.

Fig. 1.

Induction of Fpg- and UvrABC-sensitive sites by UVA irradiation in melanocytes (MCs) and normal human skin fibroblasts (NHSFs; CRL2097). Exponentially growing cells were irradiated with UVA (0, 2, and 10 J/cm²) and the genomic DNA was immediately isolated from these cells after irradiation. As positive controls, NHSF genomic DNAs were treated with H₂O₂ (10 mM for 2 h at room temperature) or irradiated with UVC (20 J/m²). The isolated genomic DNA was then reacted with (A) Fpg or (B) UvrABC, denatured and separated by electrophoresis in a 0.5% agarose gel in TBE buffer. Note: Fpg treatment greatly reduced the amount of full-size genomic DNA isolated from UVA (10 J/cm²) irradiated-HEMn-DP and -HEMn-LP but not the amount of full-size genomic DNA isolated from the same UVA-irradiated CRL2097, indicating that UVA induced Fpg-sensitive sites in HEMn-DP and HEMn-LP but not in CRL2097.

Melanocytes Have a Lower Level of HCR for H₂O₂-Damaged and UVC-Irradiated Luciferase Gene.

Because melanin is able to absorb sunlight and scavenge free radicals, it shields cells from the damaging effects of sunlight, including the induction of photoproducts and ROS (22). Results in Fig. 1 indicate that UVA irradiation of MCs generates ROS and induces ODD. Furthermore, although both epidemiological studies and animal models have shown that UVB irradiation contributes greatly to melanomagenesis (4, 29), melanoma occurs not only in tissues exposed to sunlight but also in tissues never exposed to sunlight (19–21). One possible mechanism for these disparate observations is that MCs have a reduced repair capacity for both ODD and UV photoproducts. To test this possibility, we determined the repair capacity of MCs using the HCR assay and an in vitro DNA damage-induced repair synthesis assay. The results in Fig. 2A show that the capacity of MCs to reactivate the UVC-damaged luciferase gene is significantly lower than the capacity of NHSFs, but significantly higher than that of XPA cells. We then calculated the number of photoproducts (CPDs and <6–4 > PPs) induced by different doses of UVC radiation according to the method previously described (30, 31). If we assume that 1 photoproduct in the luciferase gene is sufficient to block the expression of this gene, then the results in Fig. 2A suggest that 2 photoproducts in the coding strand of the luciferase gene are needed to block 63% of luciferase gene expression in XPA cells, which indicates XPA cells are totally defective in CPD and <6–4 > PP repair. In contrast, 9 photoproducts are needed in the luciferase gene in darkly pigmented MCs to induce the same level of reduction in luciferase gene expression, 15 in lightly pigmented MCs, and 34 in NHSFs. These results validate the assay system for measuring the repair capacity of the host cells and demonstrate that MCs are partially defective in photoproduct repair. We also observed that the lightly pigmented MCs have a lower repair capacity for photoproducts than darkly pigmented MCs. In addition, our results show that the amount of melanin in the lightly pigmented MCs was indeed lower than the darkly pigmented MCs; however, the ROS level in the former was significantly higher than in the latter (Figs. S2 and S3). It has been suggested that the ROS level in MCs depends on an interplay between the melanin-enhanced generation of ROS (15–17) and its ability to scavenge the ROS (22). The luciferase HCR assay is based on the expression level of the luciferase gene being inversely proportional to the level of DNA damage in this gene (Fig. S4). It is conceivable that the transcription-coupled repair (TCR) mechanism may affect the outcome of this assay. It has been found that TCR plays an important role in CPD repair (32, 33). However, it is generally believed that TCR does not play a role in H₂O₂-induced DNA damage (34). The disparate results of these two assays for UV-induced DNA damage between these two types of MCs, raise the possibility that TCR for UV damage is defective in lightly pigmented, but not darkly pigmented, MCs.

Fig. 2.

Determination of the capacity of NHSF, MC, XPA, XPC, and XPG cells in the repair of DNA damaged by (A) UVC irradiation, (B) H₂O₂ modifications, and (C) low concentrations of H₂O₂ by HCR. Exponentially growing cells were transfected with pGL3-luciferase plasmid modified with different doses of UVC or concentrations of H₂O₂ and unmodified pSV-β-galactosidase plasmids using Fugene 6 and the transfected cells were further incubated in growth medium for 16 h. Cell lysates were then prepared and the luciferase and β-galactosidase activities were measured. The relative repair capacity of cells was calculated as the percentage of the relative luciferase activity of the damaged plasmids vs. undamaged control. The bar represents the range of the experimental results and the position of the symbol represents the average value. The data are from three independent experiments.

The results in Fig. 2B show that the capacity of MCs to reactivate a H₂O₂- modified luciferase gene is significantly lower than the capacity of NHSFs. Darkly pigmented MCs have a lower repair capacity than lightly pigmented MCs. It appears that the reduction of the expression of the H₂O₂-modified luciferase gene is biphasic in NHSFs, indicating that there are two pathways for repair of H₂O₂-induced DNA damage. The differences between NHSFs and MCs are present only at the initial phase for low levels of H₂O₂-induced DNA damage. The sharp slope at the initial phase indicates that melanin affects the major repair pathway for ODD. The results in Fig. 2 B and C show that XPA, XPC, and XPG cells have a repair capacity comparable to that of NHSFs for low levels of DNA damage induced by H₂O₂ modifications but are deficient in repair of higher levels of H₂O₂ modification. The results suggest that XPA, XPC, and XPG cells are deficient in a repair pathway operating on DNA damage induced by high levels of H₂O₂ modification.

Cell Lysates of MCs Have a Lower Capacity to Mediate DNA Damage-Induced Repair Synthesis.

The HCR assay of the damaged luciferase gene measures the repair capacity for the transcriptionally active luciferase gene. This may or may not represent the host cell repair capacity for the overall genome, which would also include the nontranscribed strand and noncoding genomic regions. To resolve this issue, we measured the capacity of cell lysates to carry out DNA damage-induced repair synthesis. We found that the level of in vitro DNA repair synthesis is proportional to the amount of UV photoproducts or ODD in the DNA substrates, which validates the use of this system for measuring the DNA repair capacity (Fig. S5). The results in Fig. 3 show that MC cell lysates have a lower capacity to carry out both photoproduct- and ODD-induced repair synthesis than NHSF cell lysates. Darkly pigmented MC lysates have a lower capability for repair synthesis induced by both UV photoproducts and ODD than lightly pigmented MC cell lysates. The results in Fig. 3 also show that cell lysates from XPA cells have the lowest capacity for repair synthesis induced by either type of DNA damage. These results suggest that the XPA protein is involved in repair of ODD as well UV photoproducts.

Fig. 3.

Determination of the capacity of cell lysates isolated from NHSF, MC, and XPA cells in mediating DNA damage-induced repair synthesis. Cell lysates were isolated from exponentially growing cells and used to mediate repair synthesis using (A) UV-damaged or (B) H₂O₂-modified pUC18 plasmid and undamaged control pBR322 plasmid as substrates in the presence of α-³²P dATP. The resultant plasmid DNAs were purified, digested with HindIII, and separated in a 1% agarose gel by electrophoresis. In the Top panel are typical photographs of ethidium bromide-stained gels, and in the Middle panel are the autoradiographs of the same gels. The relative repair capacity was calculated based on the ratio of the amount of repair synthesis over the amount of substrate DNA (Bottom). Symbols are the same as in Figs. 1 and 2. UVC dose: 1,500 J/m², H₂O₂ modification: 100 mM for 30 min at 37 °C. The data are from three independent experiments. P values for HEMn-DP vs. HEMn-LP are 0.03 for UVC-induced DNA damage and 0.09 for H₂O₂-induced DNA damage.

Melanin Interferes with in Vitro DNA Damage Specific-Repair Synthesis.

There are two possibilities to account for the reduced HCR capacity in MCs and the reduction in DNA damage-induced repair synthesis in MC cell lysates: (i) melanin directly interferes with excision repair, and/or (ii) melanin suppresses the expression of repair genes. To test the first possibility, the effect of melanin on in vitro DNA damage-induced repair synthesis was determined. Different amounts of melanin were added directly to cell lysates isolated from NHSFs and the capacity for DNA repair synthesis in these cell lysates was then determined. Results in Fig. 4 show that the addition of melanin reduced repair synthesis induced by both photoproducts and ODD; moreover, the extent of repair reduction was proportional to the amount of melanin added to the cell lysates.

Fig. 4.

Effect of melanin on DNA damage-induced repair synthesis mediated by NHSF cell lysates. DNA damage-induced repair synthesis was carried out by NHSF cell lysates (six lanes from left in A and B), the same as described in Fig. 3, except different amounts of melanin were added to the NHSF cell lysates. In A, UVC (1,500 J/m²)-irradiated DNA was used as the substrate, and in B, H₂O₂- (100 mM, 30 min at 37°C) modified DNA was used as the substrate. In HEMn-LP lane, the repair DNA synthesis was carried out by cell lysates of lightly pigmented melanocytes. The symbols are the same as in Fig. 3. The quantitative data in C are from three independent experiments.

To determine how melanin interferes with DNA repair, different amounts of melanin were added to UVC-irradiated or H₂O₂-modified NHSF genomic DNA and these DNA-melanin mixtures were then tested for their sensitivity toward the NER enzyme UvrABC and the base excision repair (BER) enzyme Fpg. The results shown in Fig. 5 reveal that the addition of melanin to the UVC-irradiated or H₂O₂-modified DNA indeed reduced its sensitivity toward UvrABC or Fpg incision, respectively. During genomic DNA isolation from MCs, it was found that melanins copurified with genomic DNAs and the color of the genomic DNAs was dark. These darkly colored genomic DNAs were resistant to UvrABC and Fpg. However, when genomic DNAs were separated from melanin–DNA complexes by electrophoresis, the genomic DNAs became sensitive to UvrABC and Fpg incision (Fig. S6). These results strongly suggest that melanin binding renders the DNA refractory to recognition and incision by NER and BER enzymes.

Fig. 5.

Effect of melanin on Fpg and UvrABC incision activity. Genomic DNA (1 μg) isolated from NHSF cells was modified with H₂O₂ (10 mM, 2 h at room temperature) or irradiated with UVC (20 J/m²), incubated with different amounts of melanin (0, 5, 20, 100, 200, and 400 ng) in TE buffer (final volume 100 μL) for 2 min and then reacted with (A) Fpg for H₂O₂-modified DNA or (B) UvrABC for UVC-irradiated DNA. The Fpg and UvrABC reaction conditions and the separation of the resultant DNAs were the same as described in Fig. 1.

UVC-Irradiated or H₂O₂-Modified supF Gene Has a Higher Mutation Rate in MCs Than in NHSFs.

Results from both HCR and in vitro repair synthesis assays indicate that MCs have a lower repair capacity for UV photoproducts and ODD. These results raise the possibility that MCs are more susceptible to DNA damage-induced mutagenesis. To date no mutation assay has been established to measure DNA damaging agent-induced mutagenesis in normal human melanocytes. Therefore, a human–Escherichia coli shuttle vector system was used to determine the effects of reduced repair capacity on DNA damaging agent-induced mutagenesis. Plasmid DNA containing supF was modified with H₂O₂ (100 mM, 30 min at 37 °C) or irradiated with UVC (1,500 J/m²) and then transfected into MCs. After 72 h of incubation, the replicated plasmid DNAs were isolated and the mutations in the supF gene determined using indicator E. coli cells. The results in Table 1 show that the H₂O₂-modified plasmid DNA induces a 7- to 19-fold higher mutation frequency in MCs than in NHSFs, and UVC-irradiated plasmid DNA induces a 7- to 14-fold higher mutation frequency in MCs than in NHSFs. Both H₂O₂- and UVC-induced DNA damage induce a higher mutation frequency in darkly pigmented MCs than in lightly pigmented MCs. The results also show that H₂O₂-modified and UVC-irradiated plasmid DNAs induce a 5-fold and a 52-fold higher mutation frequency, respectively, in XPA cells than in NHSFs. These results are consistent with the findings that demonstrated that MC and XPA cells are defective in ODD and UV photoproduct repair and that darkly pigmented MCs are more defective in these repair pathways than lightly pigmented MCs.

View this table:

Table 1.

Mutagenicity of H₂O₂- and UVC-induced damage in the supF gene in NHSF (CRL2097), MC (HEMn-LP and HEMn-DP) and XPA cells

Discussion

Skin cancers, including cutaneous melanoma, occur mainly in body regions exposed to sunlight, clearly indicating that sunlight-induced DNA damage plays a major role in skin carcinogenesis (1–6). It is generally accepted that sunlight in the UV range of 290–400 nm is the primary cause of the DNA damage that consequently triggers mutagenesis and carcinogenesis (10, 11, 29). Although shorter wavelength (290–320 nm, UVB) irradiation can induce the formation of CPDs and <6–4 > PPs in DNA, the yield of these photoproducts in the UVA region (320–400 nm) is likely very low, if not zero (12). Therefore, the role of UVA in skin carcinogenesis, particularly in melanomagenesis, remains controversial. Because skin cells absorb far more energy from UVA than UVB and UVA can penetrate deeper than UVB (35), it is imperative to understand the biochemical effects of UVA irradiation to better assess risk and to design effective measures for skin cancer prevention.

UVA irradiation has been shown to induce free radicals, including ROS, and melanin enhances this effect (15–17). Moreover, it is well established that OS induces LPO and that the byproducts of LPO—such as HNE, MDA, and Acr— inhibit DNA repair (23–25). Consistent with these results, we found that UVA irradiation induces OS and ODD in MCs (Fig. 1), MCs have a lower repair capacity for UV photoproducts and ODD (Fig. 2), and ODD and UV photoproducts induce more mutations in MCs than in NHSFs (Table 1). Together, these results lead us to propose that UVA-induced, melanin-augmented ODD in MCs and the inherently reduced repair capacity in these cells are the two key factors that contribute to cutaneous melanomagenesis. It is worth noting that mucosal melanoma occurs in regions rarely exposed to sunlight; i.e., the colon, genital organs, and the nasal septum (19–21); these particular melanomas most likely are not induced by UV photoproducts. Our results suggest that the inherently low repair capacity in MCs may contribute to melanomagenesis in these tissues.

Melanoma incidence in XP patients is 1,000-fold higher than in normal individuals (13). It is well established that XP cells are sensitive to UVC irradiation-induced cell killing and that XP gene products recognize and repair UV photoproducts and bulky chemical DNA damage. In contrast, compared to normal human fibroblasts, XP cells are not sensitive to ionizing radiation-induced cell killing (14). Therefore, it is believed that XP proteins are involved in NER, but not BER (14). Because ample epidemiological studies have indicated that UVA irradiation is related to melanoma incidence, to account for the high incidence of melanoma in XP patients it has been proposed that melanin sensitizes cells to UVA irradiation, resulting in the generation of “bulky” photoproducts, which are repaired by the NER mechanism. Because XP patients are deficient in the repair of these bulky photoproducts, XP individuals are more prone to develop melanoma. Our results show that XPA, XPC, and XPG cells are deficient in the repair of both bulky photoproducts and ODD and that both types of DNA damage induce more mutations in XPA cells than in NHSF cells (Fig. 2 and Table 1). These results indicate that the XPA gene product is involved in both NER and BER mechanisms. Furthermore, our results show that UVA irradiation does not induce bulky photoproducts. Our findings provide a plausible explanation for why XP patients are more prone to develop melanoma: UVA irradiation induces more ODD and OS in MCs than in fibroblasts and XP individuals are deficient in ODD repair.

It is intriguing that, compared to NHSFs, MCs have lower BER and NER capacity; the underlying mechanisms for this phenomenon are unclear. Previously, we have found that OS and LPO byproducts, such as 4-HNE, MDA, and Acr, can cause an inhibitory effect on NER (23–25). Because melanin is a known ROS scavenger, it is unlikely that without UVA irradiation, MCs would have a higher level of ROS than NHSFs. In fact, we found that the ROS level in MCs is similar to that in NHSFs. We also found that the addition of melanin to NHSF lysates directly causes an inhibitory effect on repair synthesis and that melanin-associated DNA is refractory to repair enzymes. Hence, it is possible that the inhibitory effect of melanin on DNA repair arises through the interaction of melanin with repair proteins directly and/or with DNA. Because both scenarios could occur in vivo, we propose that reduced DNA repair capacity contributes to melanomagenesis, particularly in mucosal melanomas.

Materials and Methods

Culture of Primary Human Melanocytes and Skin Fibroblasts.

Human epidermal melanocytes lightly pigmented (HEMn-LP) and human epidermal melanocytes darkly pigmented (HEMn-DP) were maintained in Medium 254 supplemented with human melanocyte growth supplement (Cascade Biologics, Invitrogen). The amount of melanin in each type of cell was checked by the melanin assay (Fig. S2) (36). Primary culture of normal human skin fibroblasts (NHSFs, CRL2097, American Type Culture Collection) was grown in minimum essential medium (Invitrogen) supplemented with 10% FBS. Human NER-deficient XPA (GM05509), XPC (GM01736), and XPG fibroblasts (GM16398) (National Institute of General Medical Sciences, Human Genetic Cell Repository) were grown in minimum essential medium supplemented with 15% FBS.

UVA Irradiation and Genomic DNA Isolation.

The monolayer ~70% confluent cells were irradiated with different doses (0, 2, and 10 J/cm²) of UVA light (LT18W/009UV) filtered through Mylar polyester film (Du Pont) in PBS buffer. Immediately after irradiation, cells were harvested and the genomic DNA was isolated as previously described (23).

Fpg and UvrABC Incision Assays.

Fpg, UvrA, UvrB, and UvrC were isolated from E. coli cells as previously described (37, 38). It is well established that the three UvrABC proteins work in concert to incise the UVC photoproducts CPD and pyrimidine < 6–4 > pyrimidone (27, 28), and Fpg can incise oxidative base damage such as 8-oxo-deoxyguanosine (8-oxo-dG) and its open-ring products (26). The Fpg incision assay was carried out in the same manner as described by Boiteux et al. (38). The UvrABC incision assay and the separation of the resultant DNA were carried out as previously described (23).

Host Cell Reactivation, in Vitro Repair Synthesis, and supF Mutation Assays.

Methods for isolation of plasmid luciferase plasmid pGL3, pSV-β-galactosidase, pUC18, pBR322, and pSP189, the HCR assay, the in vitro repair synthesis assay, and supF mutation detection were the same as previously described (23–25).

Acknowledgments

We thank Ms. Josephine Kuo for technical assistance and Ms. Neva Setlow, a guest appointee at the Brookhaven National Laboratory, for assistance with tissue culture. We also thank Drs. Richard Setlow and Michael Patrick, Judith Zelikoff, and Yen-Yee Nydam for critical review of this manuscript. This work was supported by National Institutes of Health Grants (CA114541, ES014641, CA99007, and ES00260).

Footnotes

²To whom correspondence should be addressed. E-mail: moon-shong.tang{at}nyumc.org.
Author contributions: H.-T.W., B.C., and M.-s.T. designed research; H.-T.W. and B.C. performed research; H.-T.W., B.C., and M.-s.T. analyzed data; and H.-T.W., B.C., and M.-s.T. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1005244107/-/DCSupplemental.

Freely available online through the PNAS open access option.

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OpenUrl CrossRef PubMed
↵
1. Bustamante J,
2. Bredeston L,
3. Malanga G,
4. Mordoh J
(1993) Role of melanin as a scavenger of active oxygen species. Pigment Cell Res 6:348–353.
OpenUrl CrossRef PubMed
↵
1. Feng Z,
2. Hu W,
3. Hu Y,
4. Tang MS
(2006) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair. Proc Natl Acad Sci USA 103:15404–15409.
OpenUrl Abstract/FREE Full Text
↵
1. Feng Z,
2. Hu W,
3. Marnett LJ,
4. Tang MS
(2006) Malondialdehyde, a major endogenous lipid peroxidation product, sensitizes human cells to UV- and BPDE-induced killing and mutagenesis through inhibition of nucleotide excision repair. Mutat Res 601:125–136.
OpenUrl PubMed
↵
1. Feng Z,
2. Hu W,
3. Tang MS
(2004) Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: A possible mechanism for lipid peroxidation-induced carcinogenesis. Proc Natl Acad Sci USA 101:8598–8602.
OpenUrl Abstract/FREE Full Text
↵
1. Tchou J,
2. et al.
(1994) Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA. J Biol Chem 269:15318–15324.
OpenUrl Abstract/FREE Full Text
↵
1. Vreeswijk MP,
2. et al.
(1994) Analysis of repair of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts in transcriptionally active and inactive genes in Chinese hamster cells. J Biol Chem 269:31858–31863.
OpenUrl Abstract/FREE Full Text
↵
1. Pfeifer G
1. Tang M
(1996) in Technologies for Detection of DNA Damage and Mutations, ed Pfeifer G (Plenum, New York), pp 139–153.
↵
1. De Fabo EC,
2. Noonan FP,
3. Fears T,
4. Merlino G
(2004) Ultraviolet B but not ultraviolet A radiation initiates melanoma. Cancer Res 64:6372–6376.
OpenUrl Abstract/FREE Full Text
↵
1. Tang M,
2. Lieberman MW,
3. King CM
(1982) uvr genes function differently in repair of acetylaminofluorene and aminofluorene DNA adducts. Nature 299:646–648.
OpenUrl CrossRef PubMed
↵
1. Friedberg E,
2. et al.
(2006) DNA Repair and Mutagenesis (ASM, Washington, DC), pp 9–69.
↵
1. Mellon I,
2. Spivak G,
3. Hanawalt PC
(1987) Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51:241–249.
OpenUrl CrossRef PubMed
↵
1. Siede W,
2. Kow YW,
3. Doetsch PW
1. Tang M
(2006) in DNA Damage Recognition, eds Siede W, Kow YW, Doetsch PW (Taylor & Francis, New York), pp 181–199.
↵
1. Thorslund T,
2. Sunesen M,
3. Bohr VA,
4. Stevnsner T
(2002) Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias. DNA Repair (Amst) 1:262–273.
OpenUrl
↵
1. Bruls WA,
2. Slaper H,
3. ven der Leun JC,
4. Berrens L
(1984) Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 40:485–494.
OpenUrl PubMed
↵
1. Price K,
2. Linge C
(1999) The presence of melanin in genomic DNA isolated from pigmented cell lines interferes with successful polymerase chain reaction: A solution. Melanoma Res 9:5–9.
OpenUrl PubMed
↵
1. Thomas DC,
2. Levy M,
3. Sancar A
(1985) Amplification and purification of UvrA, UvrB, and UvrC proteins of Escherichia coli. J Biol Chem 260:9875–9883.
OpenUrl Abstract/FREE Full Text
↵
1. Boiteux S,
2. O'Connor TR,
3. Lederer F,
4. Gouyette A,
5. Laval J
(1990) Homogeneous Escherichia coli FPG protein. A DNA glycosylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites. J Biol Chem 265:3916–3922.
OpenUrl Abstract/FREE Full Text

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Genetics

Proceedings of the National Academy of Sciences: 107 (27)

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[1] ↵
Brash DE
(1997) Sunlight and the onset of skin cancer. Trends Genet 13:410–414.
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[24] Setlow RB,

[25] Grist E,

[26] Thompson K,

[27] Woodhead AD

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Dudek J,
Merlino G,
De Fabo EC
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[29] Noonan FP,

[30] Dudek J,

[31] Merlino G,

[32] De Fabo EC

[33] ↵
Pfeifer GP,
You YH,
Besaratinia A
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[35] You YH,

[36] Besaratinia A

[37] ↵
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Sage E,
Douki T
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[43] et al.

[44] ↵
Kraemer KH,
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Andrews AD,
Lambert WC
(1994) The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer. The xeroderma pigmentosum paradigm. Arch Dermatol 130:1018–1021.
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[46] Lee MM,

[47] Andrews AD,

[48] Lambert WC

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[50] Friedberg E,

[51] et al.

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Young AR
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[54] Young AR

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Kvam E,
Dahle J
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[57] Dahle J

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Tyrrell RM
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[60] Tyrrell RM

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[65] Madison JF,

[66] Hwu WJ,

[67] Bayar S,

[68] Salem RR

[69] ↵
Patrick RJ,
Fenske NA,
Messina JL
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[70] Patrick RJ,

[71] Fenske NA,

[72] Messina JL

[73] ↵
Thompson LD,
Wieneke JA,
Miettinen M
(2003) Sinonasal tract and nasopharyngeal melanomas: A clinicopathologic study of 115 cases with a proposed staging system. Am J Surg Pathol 27:594–611.
OpenUrl CrossRef PubMed

[74] Thompson LD,

[75] Wieneke JA,

[76] Miettinen M

[77] ↵
Bustamante J,
Bredeston L,
Malanga G,
Mordoh J
(1993) Role of melanin as a scavenger of active oxygen species. Pigment Cell Res 6:348–353.
OpenUrl CrossRef PubMed

[78] Bustamante J,

[79] Bredeston L,

[80] Malanga G,

[81] Mordoh J

[82] ↵
Feng Z,
Hu W,
Hu Y,
Tang MS
(2006) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair. Proc Natl Acad Sci USA 103:15404–15409.
OpenUrl Abstract/FREE Full Text

[83] Feng Z,

[84] Hu W,

[85] Hu Y,

[86] Tang MS

[87] ↵
Feng Z,
Hu W,
Marnett LJ,
Tang MS
(2006) Malondialdehyde, a major endogenous lipid peroxidation product, sensitizes human cells to UV- and BPDE-induced killing and mutagenesis through inhibition of nucleotide excision repair. Mutat Res 601:125–136.
OpenUrl PubMed

[88] Feng Z,

[89] Hu W,

[90] Marnett LJ,

[91] Tang MS

[92] ↵
Feng Z,
Hu W,
Tang MS
(2004) Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: A possible mechanism for lipid peroxidation-induced carcinogenesis. Proc Natl Acad Sci USA 101:8598–8602.
OpenUrl Abstract/FREE Full Text

[93] Feng Z,

[94] Hu W,

[95] Tang MS

[96] ↵
Tchou J,
et al.
(1994) Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA. J Biol Chem 269:15318–15324.
OpenUrl Abstract/FREE Full Text

[97] Tchou J,

[98] et al.

[99] ↵
Vreeswijk MP,
et al.
(1994) Analysis of repair of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts in transcriptionally active and inactive genes in Chinese hamster cells. J Biol Chem 269:31858–31863.
OpenUrl Abstract/FREE Full Text

[100] Vreeswijk MP,

[101] et al.

[102] ↵
Pfeifer G
Tang M
(1996) in Technologies for Detection of DNA Damage and Mutations, ed Pfeifer G (Plenum, New York), pp 139–153.

[103] Pfeifer G

[104] Tang M

[105] ↵
De Fabo EC,
Noonan FP,
Fears T,
Merlino G
(2004) Ultraviolet B but not ultraviolet A radiation initiates melanoma. Cancer Res 64:6372–6376.
OpenUrl Abstract/FREE Full Text

[106] De Fabo EC,

[107] Noonan FP,

[108] Fears T,

[109] Merlino G

[110] ↵
Tang M,
Lieberman MW,
King CM
(1982) uvr genes function differently in repair of acetylaminofluorene and aminofluorene DNA adducts. Nature 299:646–648.
OpenUrl CrossRef PubMed

[111] Tang M,

[112] Lieberman MW,

[113] King CM

[114] ↵
Friedberg E,
et al.
(2006) DNA Repair and Mutagenesis (ASM, Washington, DC), pp 9–69.

[115] Friedberg E,

[116] et al.

[117] ↵
Mellon I,
Spivak G,
Hanawalt PC
(1987) Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51:241–249.
OpenUrl CrossRef PubMed

[118] Mellon I,

[119] Spivak G,

[120] Hanawalt PC

[121] ↵
Siede W,
Kow YW,
Doetsch PW
Tang M
(2006) in DNA Damage Recognition, eds Siede W, Kow YW, Doetsch PW (Taylor & Francis, New York), pp 181–199.

[122] Siede W,

[123] Kow YW,

[124] Doetsch PW

[125] Tang M

[126] ↵
Thorslund T,
Sunesen M,
Bohr VA,
Stevnsner T
(2002) Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias. DNA Repair (Amst) 1:262–273.
OpenUrl

[127] Thorslund T,

[128] Sunesen M,

[129] Bohr VA,

[130] Stevnsner T

[131] ↵
Bruls WA,
Slaper H,
ven der Leun JC,
Berrens L
(1984) Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 40:485–494.
OpenUrl PubMed

[132] Bruls WA,

[133] Slaper H,

[134] ven der Leun JC,

[135] Berrens L

[136] ↵
Price K,
Linge C
(1999) The presence of melanin in genomic DNA isolated from pigmented cell lines interferes with successful polymerase chain reaction: A solution. Melanoma Res 9:5–9.
OpenUrl PubMed

[137] Price K,

[138] Linge C

[139] ↵
Thomas DC,
Levy M,
Sancar A
(1985) Amplification and purification of UvrA, UvrB, and UvrC proteins of Escherichia coli. J Biol Chem 260:9875–9883.
OpenUrl Abstract/FREE Full Text

[140] Thomas DC,

[141] Levy M,

[142] Sancar A

[143] ↵
Boiteux S,
O'Connor TR,
Lederer F,
Gouyette A,
Laval J
(1990) Homogeneous Escherichia coli FPG protein. A DNA glycosylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites. J Biol Chem 265:3916–3922.
OpenUrl Abstract/FREE Full Text

[144] Boiteux S,

[145] O'Connor TR,

[146] Lederer F,

[147] Gouyette A,

[148] Laval J

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Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts

Abstract

Results

Oxidative DNA Damage and Bulky Photoproducts Induced by UVA Irradiation in MCs.

Melanocytes Have a Lower Level of HCR for H₂O₂-Damaged and UVC-Irradiated Luciferase Gene.

Cell Lysates of MCs Have a Lower Capacity to Mediate DNA Damage-Induced Repair Synthesis.

Melanin Interferes with in Vitro DNA Damage Specific-Repair Synthesis.

UVC-Irradiated or H₂O₂-Modified supF Gene Has a Higher Mutation Rate in MCs Than in NHSFs.

Discussion

Materials and Methods

Culture of Primary Human Melanocytes and Skin Fibroblasts.

UVA Irradiation and Genomic DNA Isolation.

Fpg and UvrABC Incision Assays.

Host Cell Reactivation, in Vitro Repair Synthesis, and supF Mutation Assays.

Acknowledgments

Footnotes

References

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Abstract

Results

Oxidative DNA Damage and Bulky Photoproducts Induced by UVA Irradiation in MCs.

Melanocytes Have a Lower Level of HCR for H2O2-Damaged and UVC-Irradiated Luciferase Gene.

Cell Lysates of MCs Have a Lower Capacity to Mediate DNA Damage-Induced Repair Synthesis.

Melanin Interferes with in Vitro DNA Damage Specific-Repair Synthesis.

UVC-Irradiated or H2O2-Modified supF Gene Has a Higher Mutation Rate in MCs Than in NHSFs.

Discussion

Materials and Methods

Culture of Primary Human Melanocytes and Skin Fibroblasts.

UVA Irradiation and Genomic DNA Isolation.

Fpg and UvrABC Incision Assays.

Host Cell Reactivation, in Vitro Repair Synthesis, and supF Mutation Assays.

Acknowledgments

Footnotes

References

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Melanocytes Have a Lower Level of HCR for H₂O₂-Damaged and UVC-Irradiated Luciferase Gene.

UVC-Irradiated or H₂O₂-Modified supF Gene Has a Higher Mutation Rate in MCs Than in NHSFs.