Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells. Possible role of Dewar photoproducts in solar mutagenesis.
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In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0. 12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4)PP photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis. (+info)
Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase eta.
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Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase eta, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated approximately 4-fold during cell proliferation. These results suggest that DNA polymerase eta plays a role in DNA replication, though the enzyme is not essential for viability. (+info)
Mechanisms and implications of the age-associated decrease in DNA repair capacity.
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Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process. (+info)
Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis.
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UV-damaged DNA-binding activity (UV-DDB) is deficient in some xeroderma pigmentosum group E individuals due to mutation of the p48 gene, but its role in DNA repair has been obscure. We found that UV-DDB is also deficient in cell lines and primary tissues from rodents. Transfection of p48 conferred UV-DDB to hamster cells, and enhanced removal of cyclobutane pyrimidine dimers (CPDs) from genomic DNA and from the nontranscribed strand of an expressed gene. Expression of p48 suppressed UV-induced mutations arising from the nontranscribed strand, but had no effect on cellular UV sensitivity. These results define the role of p48 in DNA repair, demonstrate the importance of CPDs in mutagenesis, and suggest how rodent models can be improved to better reflect cancer susceptibility in humans. (+info)
Protection by ultraviolet A and B sunscreens against in situ dipyrimidine photolesions in human epidermis is comparable to protection against sunburn.
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Sunscreens prevent sunburn and may also prevent skin cancer by protecting from ultraviolet-induced DNA damage. We assessed the ability of two sunscreens, with different spectral profiles, to inhibit DNA photodamage in human epidermis in situ. One formulation contained the established ultraviolet B filter octyl methoxycinnamate, whereas the other contained terephthalylidene dicamphor sulfonic acid, a new ultraviolet A filter. Both formulations had sun protection factors of 4 when assessed with solar simulating radiation in volunteers of skin type I/II. We tested the hypothesis that sun protection factors would indicate the level of protection against DNA photodamage. Thus, we exposed sunscreen-treated sites to four times the minimal erythema dose of solar simulating radiation, whereas vehicle and control sites were exposed to one minimal erythema dose. We used monoclonal antibodies against thymine dimers and 6-4 photoproducts and image analysis to quantify DNA damage in skin sections. A dose of four times the minimal erythema dose, with either sunscreen, resulted in comparable levels of thymine dimers and 6-4 photoproducts to one minimal erythema dose +/- vehicle, providing evidence that the DNA protection factor is comparable to the sun protection factor. The lack of difference between the sunscreens indicates similar action spectra for erythema and DNA photodamage and that erythema is a clinical surrogate for DNA photodamage that may lead to skin cancer. (+info)
Levels and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in skin of sporadic basal cell carcinoma patients.
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The 32P-postlabeling method was applied to measure directly the levels and repair rates of specific cyclobutane pyrimidine dimers and 6-4 photoproducts in 10 basal cell carcinoma patients and 10 controls matched on age, skin type, and gender after exposure to 400 J per m2 of solar simulating radiation on previously unexposed buttock skin. The results showed an identical level of photoproducts at 0 h after solar simulating radiation in the basal cell carcinoma group and the control group. Erythemal response correlated with the repair of cyclobutane pyrimidine dimers within 24 h in both groups, i.e., repair was faster in those with a strong erythemal reaction. The basal cell carcinoma patients showed a somewhat slower repair of photoproducts in skin compared with the controls, but the result was not significant. Photoproducts formed at the TTC sites were repaired faster than those at the TTT sites for both cyclobutane pyrimidine dimers and 6-4 photoproducts in the basal cell carcinoma group and in the controls. (+info)
The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin.
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We and colleagues have suggested that deletions of mitochondrial DNA may be useful as a biomarker of ultraviolet radiation exposure in skin. In this study using a southwestern approach involving monoclonal antibodies against thymine dimers we provide direct evidence for the presence of ultraviolet-induced damage in mitochondrial DNA purified from any nuclear DNA contamination. Previous studies have been limited, as they have focused on the frequency of a single mitochondrial DNA deletion. Therefore we have addressed the question of the spectrum of mitochondrial DNA deletions in skin and whether this can be used as an index of overall DNA damage. We have used a long polymerase chain reaction technique to determine the mitochondrial DNA deletion spectrum of almost the entire mitochondrial genome in 71 split skin samples in relation to sun exposure. There was a significant increase in the number of deletions with increasing ultraviolet exposure in the epidermis (Kruskal-Wallis test, p = 0.0015) but not the dermis (p = 0.6376). The findings in the epidermis are not confounded by any age-dependent increases in mitochondrial DNA deletions also detected by the long polymerase chain reaction technique. The large spectrum of deletions identified in our study highlights the ubiquitous nature and the high mutational load of mitochondrial DNA associated with ultraviolet exposure and chronologic aging. Compared with the detection of single deletions using competitive polymerase chain reaction, we show that long polymerase chain reaction is a sensitive technique and may therefore provide a more comprehensive, although not quantitative, index of overall mitochondrial DNA damage in skin. (+info)
Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase. (+info)