Ultraviolet Photoproducts Research Articles

Validation of Phage T7 Biological Dosimeter by Quantitative Polymerase Chain Reaction Using Short and Long Segments of Phage T7 DNA ¶

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.

Merit Award Winners

Vol. 112, No. 14 AnnouncementsOpen AccessMerit Award Winners Published:1 October 2004https://doi.org/10.1289/ehp.112-1247589AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit AbstractThe NIEHS is pleased to announce that Raymond F. Burk, a professor of pathology and director of the Clinical Nutrition Research Unit at Vanderbilt University Medical Center, and Gerd P. Pfeifer, a professor and chair of the Division of Biology at the City of Hope Beckman Research Institute, have each received an award under the Method to Extend Research in Time (MERIT) Award Program. MERIT awards are offered to investigators who have demonstrated superior skill and outstanding productivity during the course of their previous research endeavors. MERIT awards relieve investigators from writing frequent renewal applications by providing the opportunity to gain up to 10 years of support in two segments.Burk’s research has centered on the characterization of the biological function of selenium, an essential human nutrient found in virtually all tissues, and the determination of selenoproteins responsible for that function. Early work on this grant identified and characterized selenoprotein-P (Se-P), a plasma protein made primarily in the liver that carries selenium to the brain and testis. Mice lacking the protein have male infertility and brain injury; conversely, a high-selenium diet prevents brain injury.Toxicity studies by this lab demonstrated that selenium deficiency upregulates enzymes important to the detoxification of some compounds, while the same deficiency increases the toxicity of other compounds. Burk has shown that cirrhosis is associated with low plasma Se-P and has collaborated in studies showing that selenium deficiency is not necessary for the development of colorectal adenomas. Current studies are evaluating the possibility that the Se-P delivery system to the brain can be impaired and lead to human neurodegeneration. Burk is also investigating the mechanism of transport of selenium via Se-P from mother to fetus.As a clinical physician and former director of the Clinical Nutrition Research Unit, who has received awards for his work from the American Institute of Nutrition, Burk is in a unique position to translate research conducted in animals to human clinical research.Pfeifer’s research has contributed greatly to our understanding of the molecular mechanisms associated with skin carcinogenesis in response to ultraviolet (UV) light injury and to the study of site specificity of UV photoproducts. He demonstrated that a different pattern of damage is observed when cells (as opposed to naked DNA) are irradiated with UV light, and he characterized the methylation and nucleosome locations of the p53 genes.With colleague Gerald P. Holmquist, Pfeifer applied ligation-mediated polymerase chain reaction to the study of DNA damage at the nucleotide level. This allowed the critical observation that differences in mutation frequency depend upon the efficiency of DNA repair rather than lesion induction alone. The Pfeifer lab demonstrated an important role of the DNA base 5-methylcytosine in UV damage formation and mutagenesis.Pfeifer proposes to characterize DNA-damaging and mutagenic properties of UVA irradiation, a component of the solar spectrum that has been linked to melanoma. He then will attempt to demonstrate a molecular link between sunlight exposure and melanoma. Studies will determine the in vivo roles of DNA polymerases and other proteins likely to be involved in UV mutagenesis.Pfeifer’s basic science findings are leading to a better understanding of the mechanisms of UV light and also lay the foundation for prevention and treatment of skin cancers.FiguresReferencesRelatedDetails Vol. 112, No. 14 October 2004Metrics About Article Metrics Publication History Originally published1 October 2004Published in print1 October 2004 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Nanoscale spatial induction of ultraviolet photoproducts in cellular DNA by three-photon near-infrared absorption.

The high-resolution spatial induction of ultraviolet (UV) photoproducts in mammalian cellular DNA is a goal of many scientists who study UV damage and repair. Here we describe how UV photoproducts can be induced in cellular DNA within nanometre dimensions by near-diffraction-limited 750 nm infrared laser radiation. The use of multiphoton excitation to induce highly localized DNA damage in an individual cell nucleus or mitochondrion will provide much greater resolution for studies of DNA repair dynamics and intracellular localization as well as intracellular signalling processes and cell-cell communication. The technique offers an advantage over the masking method for localized irradiation of cells, as the laser radiation can specifically target a single cell and subnuclear structures such as nucleoli, nuclear membranes or any structure that can be labelled and visualized by a fluorescent tag. It also increases the time resolution with which migration of DNA repair proteins to damage sites can be monitored. We define the characteristics of localized DNA damage induction by near-infrared radiation and suggest how it may be used for new biological investigations.

DNA mismatch repair proteins: potential guardians against genomic instability and tumorigenesis induced by ultraviolet photoproducts.

In addition to their established role in repairing post-replicative DNA errors, DNA mismatch repair proteins contribute to cell cycle arrest and apoptosis in response to a wide range of exogenous DNA damage (e.g., alkylation-induced lesions). The role of DNA mismatch repair in response to ultraviolet-induced DNA damage has been historically controversial. Recent data, however, suggest that DNA mismatch repair proteins probably do not contribute to the removal of ultraviolet-induced DNA damage, but may be important in suppressing mutagenesis, effecting apoptosis, and suppressing tumorigenesis following exposure to ultraviolet radiation.

Validation of Phage T7 Biological Dosimeter by Quantitative Polymerase Chain Reaction Using Short and Long Segments of Phage T7 DNA¶

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.

Hap1p Photofootprinting as an In Vivo Assay of Repression Mechanism in Saccharomyces cerevisiae

This chapter explores the gene expression levels, resulting from the dynamic interplay of activators and repressors. These factors influence the basal transcriptional machinery, directly or indirectly, through interactions that govern each other's activity or access to DNA. The strategy employs in vivo ultraviolet (UV) photofootprinting in order to detect activator binding with one of the most widely used yeast reporter genes, CYC1-lacZ. It has been utilized for analysis of many different repressors and repression sites. The UV photofootprinting has been widely used for analyzing the DNA structure and protein–DNA interaction in vitro and in vivo. This technique is based on the premise that, changes in DNA structure or the formation of protein–DNA complexes alter rates of dimerization of adjacent based on a particular DNA strand. The chapter also highlights that binding of Hap1p to UAS1 is detectable by in vivo UV photofootprinting. The useful feature of footprinting is to bind the Hap1p results in a positive signal, and the enhancement of formation of UV photoproducts. Thus, detection is extremely sensitive compared to a protection footprint because even limited occupancy of the UAS1 site yields a detectable signal.

Involvement of Vertebrate Polκ in Rad18-independent Postreplication Repair of UV Damage

DNA damage, which is left unrepaired by excision repair pathways, often blocks replication, leading to lesions such as breaks and gaps on the sister chromatids. These lesions may be processed by either homologous recombination (HR) repair or translesion DNA synthesis (TLS). Vertebrate Polkappa belongs to the DNA polymerase Y family, as do most TLS polymerases. However, the role for Polkappa in vertebrate cells is unclear because of the lack of reverse genetic studies. Here, we generated cells deficient in Polkappa (polkappa cells) from the chicken B lymphocyte line DT40. Although purified Polkappa is unable to bypass ultraviolet (UV) damage, polkappa cells exhibited increased UV sensitivity, and the phenotype was suppressed by expression of human and chicken Polkappa, suggesting that Polkappa is involved in TLS of UV photoproduct. Defects in both Polkappa and Rad18, which regulates TLS in yeast, in DT40 showed an additive effect on UV sensitivity. Interestingly, the level of sister chromatid exchange, which reflects HR-mediated repair, was elevated in normally cycling polkappa cells. This implies functional redundancy between HR and Polkappa in maintaining chromosomal DNA. In conclusion, vertebrate Polkappa is involved in Rad18-independent TLS of UV damage and plays a role in maintaining genomic stability.

Modulation of repair of ultraviolet damage in the host-cell reactivation assay by polymorphic XPC and XPD/ERCC2 genotypes.

DNA repair capacity (DRC) plays an important role in genetic susceptibility to cancer. Polymorphisms of a number of DNA repair genes involved in several distinct pathways have been identified. However, their effects on repair function have not been well characterized. We demonstrated previously that DRC for removal of benzo[a]pyrene diol epoxide-induced DNA damage measured by a host-cell reactivation assay was modulated by two XPD/ERCC2 polymorphisms in lung cancer. In this report, we investigated the association between the repair phenotype of ultraviolet (UV)-induced damage and genotypes of three DNA repair genes, XPC and XPD [involved in nucleotide excision repair (NER)] and XRCC1 [involved in base excision repair (BER)]. We measured DRC for removal of UV photoproducts by the host-cell reactivation assay in cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. We also typed these subjects for five polymorphisms in these three DNA repair genes (at intron 9 of XPC; exons 6, 10 and 23 of XPD and exon 10 of XRCC1). Compared with wild-type homozygotes, subjects homozygous for polymorphisms of the two NER genes consistently had suboptimal DRC. The DRC was consistently lower in subjects homozygous for XPC, XPD or both than in subjects with other genotypes, although the difference was not statistically significant for XPD variants. In contrast, the polymorphic allele of the BER gene, XRCC1, had no consistent effect on DRC. We concluded that these NER polymorphisms may modulate DRC and may be useful biomarkers for identifying individuals at risk of developing cancer.

Ultraviolet Photoproduct Levels in Melanocytic Nevi and Surrounding Epidermis in Human Skin In Situ

Melanocytic nevi are localized benign proliferations of melanocytes. The number of nevi has been shown to be the major risk marker for the development of cutaneous melanoma. This study compares the induction of photoproducts in nevi and in surrounding skin after exposure to solar-simulating radiation. Cyclobutane pyrimidine dimers (TT=T and TT=C) and 6-4 photoproducts (TT-T and TT-C) were measured in 20 nevi and 20 surrounding skin samples obtained from 14 subjects, using a 32P-postlabeling method. The amount of all four types of photoproducts in nevi was found to be 3-5-fold lower than that in surrounding skin, and the difference was statistically significant (paired t test, p < 0.01). In nevi, the photoproduct level was significantly associated with the color of nevi (the lowest level in the darkest color of nevi; r = -0.86, p < 0.01 for TT=T; r = -0.68, p < 0.01 for TT=C). Our findings suggest that the magnitude of the DNA damage is not a sole risk marker for the development of cutaneous melanoma.

Immunoexpression of Ultraviolet Photoproducts and p53 Mutation Analysis in Atypical Fibroxanthoma and Superficial Malignant Fibrous Histiocytoma

p53 mutation is one of the major results of ultraviolet (UV) radiation. UV photoproducts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6–4) photoproducts (64PPs) also play an important role in skin cancer development. Atypical fibroxanthoma (AFX), which mimics malignant fibrous histiocytoma (MFH) histologically, occurs in the sun-exposed skin of the elderly, and therefore, an association with UV has long been suspected. Eighteen fibrohistiocytic skin lesions comprising AFX (n = 7), storiform-pleomorphic type MFH centered in the subcutis (superficial MFH; S-MFH; n = 4) and benign fibrous histiocytoma (BFH; n = 7) were used for immunohistochemical and molecular analysis. Eight cases of deep MFH (D-MFH) were also analyzed for UV photoproduct expression for the purposes of comparison. Immunohistochemically, the CPD scores of AFX (3.6 ± 0.4) were significantly higher than those of S-MFH (1.3 ± 0.8), D-MFH (0.8 ± 0.5), or BHF (1.4 ± 0.7); however, the 64PP scores were extremely low in all these tumors (AFX, 0.1 ± 0.1; S-MFH, 0.0 ± 0.0; D-MFH, 0.0 ± 0.0; and BHF, 0.0 ± 0.0). AFX, S-MFH, and BFH showed immunoexpression for p53 (2/7, 2/4, and 0/7), respectively. p53 mutations were detected in AFX (4/6; 67%) and S-MFH (1/4; 25%), but not in BFH (0/5; 0%) using polymerase chain reaction–single-strand conformation polymorphism, and all of the mutations in AFX were either C-T transitions or at dipyrimidine sites. In conclusion, AFX and S-MFH are both similar fibrohistocytic lesions; however, AFX has high immunoreactivity for CPDs compared with S-MFH, D-MFH, or BFH. These data suggest that CPDs may play an important role in the pathogenesis of AFX.

In Vivo Detection of Ultraviolet Photoproducts and Their Repair in Purkinje Cells

We previously developed a highly sensitive method to assess in situ repair kinetics of ultraviolet (UV)-induced DNA photoproducts in epidermal cells using monoclonal antibodies specific for cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6–4) photoproducts (64PPs) by immunohistochemistry. In order to determine whether nucleotide excision repair capacity is operative in postmitotic mature neurons, brain surfaces of adult mice were exposed to UVB, and induction and removal of CPDs and 64PPs in Purkinje cell DNA were assessed immunohistochemically. UVB penetrated brain tissue to a depth sufficient to allow quantitative study. CPDs but not 64PPs were clearly detectable in the nuclei of Purkinje cells at doses >500 J/m2, in a dose-dependent manner. A time course experiment showed a statistically significant decrease of CPDs with time after irradiation. Although there was no apparent removal on Day 1, about half of CPDs were removed within 5 days, and the repair was essentially completed by Day 10. We conclude that non-dividing cerebellar neuronal cells can indeed repair UV-induced DNA damage, but with relatively low efficiency as compared with dividing epidermal cells.

Mitotic viability and metabolic competence in UV-irradiated yeast cells

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Δ and rad7Δ). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Δ) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Δ cells are mitotically viable after a UV dose of 12 J/m2, whereas >75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Δ cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.

Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.

Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway.

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.

TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box.

DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

P XVIII.1 - P XVIII.1 Induction of UV photoproducts and DNA damage by solar simulator UV irradiation

The recent increased incidence of skin cancer and the depletion of the ozone layer has increased interest in the ultraviolet (UV) component of natural sunlight and its role in the induction of skin cancer. Previous research on UV radiation has concentrated on UVC (254nm) but, as only UVB and UVA are present in natural sunlight, its relevance is unknown. We have investigated the induction of two forms of direct DNA damage - the pyrimidine dimer and the (6-4) photoproduct - in human DNA repair deficient XP-G (Xeroderma pigmentosum group G) lymphoblastoid cells following exposure to simulated sunlight. As exposure to natural sunlight is highly variable, a solar simulator lamp was used which is known to mimic natural sunlight at midday in Central Europe. Cells were irradiated on ice to minimise DNA repair and the relative induction of pyrimidine dimers and (6-4) photoproducts was measured using specific monoclonal antibodies and a computer assisted image analysis system. A time dependent increase in both cyclobutane dimer and (6-4) photoproduct antibody binding sites was seen. The increases in pyrimidine dimer and (6-4) photoproduct antibody binding sites differed to that reported with natural sunlight in the UK but was similar to that seen with amore » similar solar simulator lamp.« less

Detection of Ultraviolet Photoproducts in Mouse Skin Exposed to Natural Sunlight

In the present study, we for the first time investigated the formation of ultraviolet (UV) photoproducts, cyclobutane pyrimidine dimers (CPDs), pyrimidine‐pyrimidone (6–4) photoproducts (64PPs) and Dewar isomers, in vivo in shaved and depilated C3H/HeN mouse skin exposed to natural sunlight (NSL) at noon for 5 min to 1 h in mid‐summer, using a highly sensitive immunohistochemical method. This method permits the quantitative analysis of UV‐photoproducts in formalin‐fixed, paraffin‐embedded sections with specific antibodies against CPDs, 64PPs and Dewar isomers. We demonstrated that the induction of CPDs in vivo in mouse skin by NSL was exposure time‐dependent, but the accumulation of 64PPs or Dewar isomers was comparatively low in the skin sections from mice exposed to NSL in vivo. The results indicate that CPDs are the main photoproducts in vivo induced by sunlight and that their formation and repair may be important in connection with carcinogenesis in sun‐exposed areas of human skin.

Arrested DNA replication in Xenopus and release by Escherichia coli mutagenesis proteins.

Xenopus oocytes and oocyte nuclear extracts repair ultraviolet photoproducts on double-stranded (ds) DNA and replicate single-stranded (ss) to ds DNA. M13 ss DNA molecules containing cyclobutane pyrimidine dimers were maintained but not replicated in Xenopus oocytes yet were replicated in progesterone-matured oocytes. The replication arrest functioned only in cis. The replication arrest was alleviated by injection into oocytes of messenger RNAs encoding the prokaryotic mutagenesis proteins UmuD'C or MucA'B. These results may help explain how cells stabilize repair or replication events on DNA with unrepairable lesions.

A modified quantitative polymerase chain reaction assay for measuring gene-specific repair of UV photoproducts in human cells.

Methods for measuring the induction and repair of ultraviolet (UV) induced modifications in short DNA fragments are essential for the study of gene-specific DNA repair. Measurements in genomic fragments of 14 kilobases (kb) or larger can be obtained using the enzyme-sensitive site (ESS) assay introduced by Hanawalt and Bohr (Bohr et al., 1985). More recently, several assays based on variations of the polymerase chain reaction (PCR) technique have been developed which have increased sensitivity (Govan et al., 1990; Kalinowski et al., 1992; Jennerwein and Eastman, 1991), even nucleotide resolution (Pfeifer et al., 1993). However, examination of these reports indicates that the PCR based DNA repair assays lack precision (Govan et al., 1990; Kalinowski et al., 1992; Tornaletti and Pfeifer, 1994; Jennerwein and Eastman, 1991). We report here, the development of a highly precise QPCR DNA repair assay. The assay was used to measure the induction and repair of UV photoproducts in a 2.7 kb genomic fragment containing the human growth hormone (hGH) gene in SL89 (wild-type) fibroblasts. The assay was exceedingly reproducible with an overall coefficient of variation from the mean of about 2.5%. This level of precision enabled the apparent simultaneous resolution of cyclobutane dimer (CPD) and (6-4) photoproduct (6-4PP) induction and repair.

UV damage and repair mechanisms in mammalian cells.

The formation of DNA photoproducts by ultraviolet (UV) light is responsible for induction of mutations and development of skin cancer. To understand UV mutagenesis, it is important to know the mechanisms of formation and repair of these lesions. Cyclobutane pyrimidine dimers and (6-4)photoproducts are the two major classes of UV-induced DNA lesions. Their distribution along DNA sequences in vivo is strongly influenced by nucleosomes and other DNA binding proteins. Repair of UV photoproducts is dependent on the transcriptional status of the sequences to be repaired and on the chromatin environment. Sensitive techniques are now available to study repair of UV damage at the level of nucleotide resolution in mammalian cells. With the aid of in vitro systems, the entire nucleotide excision repair process has been reconstituted from purified protein components with naked DNA as a substrate. Future work will focus on the development of in vitro assays for transcription-coupled repair and repair in chromatin.