DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25+/-8.45 microm; non-smokers, 21.6+/-2.06 microm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5+/-20.34 microm; non-smokers, 79.2+/-11.59 microm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13+/-10.73 microm; non-smokers, (27.2+/-4.13 microm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease that is characterized by the extreme sensitivity of the skin to sunlight. Compared to normal individuals, XP patients have a more than 1000-fold increased risk of developing cancer on sun-exposed areas of the skin. Genetic and molecular analyses have revealed that the repair of ultraviolet (UV)-induced DNA damage is impaired in XP patients owing to mutations in genes that form part of a DNA-repair pathway known as nucleotide excision repair (NER). Two other diseases, Cockayne syndrome (CS) and the photosensitive form of trichothiodystrophy (TTD), are linked to a defect in the NER pathway. Strikingly, although CS and TTD patients are UV-sensitive, they do not develop skin cancer. The recently developed animal models that mimic the human phenotypes of XP, CS and TTD will contribute to a better understanding of the etiology of these diseases and the role of UV-induced DNA damage in the development of skin cancer.     

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.     

A kinase-independent function of c-Abl in promoting proteolytic destruction of damaged DNA binding proteins

Damaged DNA binding proteins (DDBs) play a critical role in the initial recognition of UV-damaged DNA and mediate recruitment of nucleotide excision repair factors. Previous studies identified DDB2 as a target of the CUL-4A ubiquitin ligase. However, the biochemical mechanism governing DDB proteolysis and its underlying physiological function in the removal of UV-induced DNA damage are largely unknown. Here, we report that the c-Abl nonreceptor tyrosine kinase negatively regulates the repair of UV-induced photolesions on genomic DNA. Biochemical studies revealed that c-Abl promotes CUL-4A-mediated DDB ubiquitination and degradation in a manner that does not require its tyrosine kinase activity both under normal growth conditions and following UV irradiation. Moreover, c-Abl activates DDB degradation in part by alleviating the inhibitory effect of CAND1/TIP120A on CUL-4A. These results revealed a kinase-independent function of c-Abl in a ubiquitin-proteolytic pathway that regulates the damage recognition step of nucleotide excision repair.     

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.     

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.     

DNA repair in Cockayne syndrome.

Cockayne syndrome (CS) is a rare recessive genetic disease characterized in part by premature ageing and photosensitive skin. Because of the latter characteristic, this syndrome was considered to be an example of a UV-sensitive DNA repair-defective human disorder. We demonstrated normal levels of UV-induced unscheduled DNA synthesis (UDS) in four unrelated CS patients that show hypersensitivity to both UV and Mitomycin C (MMC). At low UV exposure, CS DNA shows a dose-dependent decrease in size. By contrast, heterozygotes appear to have a threshold below which there is little change in size of single strand DNA. Immediately following UV or MMC treatment, CS DNA is deficient in high molecular weight species, but undergoes a normal transition to larger DNA during a chase interval in the presence or absence of caffeine. This suggests a defect in replication or excision repair and no defect in post-replication repair (PRR). Pulse studies performed in the presence of hydroxyurea (HU) also reveal a deficient production of large DNA, suggesting the defect is in repair. As these cells have normal UDS and normal PRR, the basis for their UV sensitivity must be distinct from that observed in xeroderma pigmentosum (XP).     

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.     

The repair of large DNA adducts in mammalian cells.

This paper describes experiments involving the measurement of DNA damage and repair after treatment with 4-nitroquinoline 1-oxide (4NQO) or aflatoxin B1 (AFB1) epoxide in a number of mammalian cell cultures primarily associated with defects in the excision repair of UV-induced DNA damage. The results with transformed derivatives of XP cells belonging to different complementation groups showed that the extent of repair of 4NQO adducts at the N2 or C8 of guanosine did not correlate to the extent of repair reported by others after UV-irradiation. An examination of 4NQO repair in rodent UV-sensitive cell lines from different ERCC groups indicated that again there was little correlation between the extent of 4NQO and UV repair. However, regardless of complementation group those mutants that were defective in the repair of pyrimidine dimers and 6,4-photoproducts did exhibit a reduced ability to repair the 4NQO N2 guanosine adduct, whereas those mutants defective in pyrimidine dimer repair alone were able to repair this lesion as normal. In all of these cell lines there was a normal capacity to repair the 4NQO C8 guanosine adduct. Less extensive experiments involving AFB1 epoxide showed an XPC-transformed cell line was able to repair 40% of lesions after 6 h, whereas only 20% of repair is seen after UV. The rodent mutant V-C4 which belongs to the same ionising radiation group as irs2, was partially defective in repairing AFB1-induced damage. These experiments highlight the fact that although there are many commonalities between the repair of UV damages and lesions classed as large DNA adducts differences clearly exist, the most striking example here being the repair of the C8 guanosine 4NQO adduct which rarely correlates with a defect in UV repair.     

Radiation enhancement of the efficiency of DNA-mediated gene transfer in CHO UV-sensitive mutants

We have employed the Chinese hamster ovary (CHO) UV-sensitive mutant cell lines, UV5 and UV20, to determine whether ionizing and ultraviolet irradiation enhance the efficiency of DNA-mediated gene transfer in cells deficient in excision repair. Confluent AA8 (wild type), UV5, and UV20 cells were transfected (via polybrene and dimethyl sulfoxide treatments) with the recombinant DNA plasmid, pSV2-gpt, trypsinized, irradiated with either X rays or ultraviolet in suspension, and then plated into flasks. After a 48-h expression time, cells were trypsinized, counted, and plated in XMAT media to select for pSV2-gpt transformation. We report that X-ray irradiation enhances gene transfer in wild-type AA8 and in both UV-sensitive cell lines. Ultraviolet irradiation enhances gene transfer in AA8 and UV20, but not in UV5. Since both UV20 and UV5 are deficient in excision repair, we suggest that ultraviolet-enhanced gene transfer may involve a postreplication repair mechanism deficient in UV5.     

Excision-repair capacity in Streptococcus pneumoniae: cloning and expression of a uvr-like gene

Although deficient in photoreactivation and some SOS-like functions, Streptococcus pneumoniae has the capacity to carry out excision repair when exposed to UV light. The repair ability and sensitivity to UV irradiation or treatment with chemical agents in the wild type and a UV-sensitive mutant strain indicate that UV-induced pyrimidine dimers might be repaired in pneumococcus by a system similar to the uvr-dependent system in Escherichia coli. A gene complementing the mutation conferring UV sensitivity of the mutant strain has been cloned. The coding region directs the synthesis of a polypeptide with a molecular weight of 78 kDa. The relationship with uvr-like protein in E. coli is discussed.     

Repair of UV-induced damage in wild-type and mutant strains of Schizophyllum commune

The basidiomycete fungus Schizophyllum commune was found to have both photo-repair and dark-repair systems for UV-induced damage. Three UV-sensitive mutants were isolated and characterized for ability to repair UV-induced damage in light and dark, and for cross-sensitivity to caffeine and methyl methanesulfonate. Two of the mutants were damaged, to different extents, in their capacity for excision repair; one of these mutants was also probably damaged in post-replication repair. The third mutant was damaged only in post-replication repair.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein.     

p53-degradation by HPV-16 E6 preferentially affects the removal of cyclobutane pyrimidine dimers from non-transcribed strand and sensitizes mammary epithelial cells to UV-irradiation

Nucleotide excision repair (NER), the most versatile and ubiquitous mechanism for DNA repair, operates to remove many types of DNA base lesions. We have studied the role of p53 function in modulating the repair of DNA damage following UV irradiation in normal and p53-compromised human mammary epithelial cells (HMEC). The effect of UV-induced DNA damage on cellular cytotoxicity and apoptosis was determined in conjunction with global, gene- and strand-specific repair. Cytotoxicity studies, using clonogenic survival and MTT assays, showed that HPV-16 E6-expressing HMEC were more UV sensitive than p53-WT cell lines. High apoptotic index obtained with p53-compromised cells was in conformity to both the low clonogenic survival and the low cellular viability. No discernible differences in the formation of initial UV-induced cyclobutane pyrimidine dimers (CPD) were observed in the cell lines of varying p53 functional status. However, the extent and the rate of damage removal from genome overall were highest for p53-WT cells. Further examination of strand-specific repair in the p53 gene revealed that the removal of CPD in the non-transcribed strand (NTS) was slower in p53-compromised cells compared to the normal p53-WT cell lines. These results suggest that loss of p53 function, in the absence of other genetic alterations, decreased both overall amount of CPD repaired and their removal rate from the genome. Additionally, normal function of p53 is required for the repair of the NTS, but not of the transcribed strand (TS) in genomic DNA in human epithelial cells. Thus, failure of quantitative removal of CPD by global genomic repair (GGR), due to loss of p53 function, causes the enhanced UV sensitivity and increased damage-induced apoptosis via a p53-independent pathway. Nevertheless, recovery of cells from UV damage requires normal p53 function and efficient GGR.