Excision Repair of Uv Radiation-Induced DNA Damage in Caenorhabditis Elegans

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts [cyclobutane dimers and (6-4) photoproducts]. Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.     

Rapid repair kinetics of pyrimidine(6-4)pyrimidone photoproducts in human cells are due to excision rather than conformational change.

UV-induced pyrimidine(6-4)pyrimidone photoproducts in DNA of mammalian cells are apparently repaired much more rapidly than cyclobutane dimers. Since only immunological assays for (6-4) photoproducts have been sensitive enough for repair measurements, it was possible that these apparently rapid repair kinetics reflected a change in physical conformation of antibody-binding sites, resulting in epitope loss rather than excision. To discriminate between these possibilities, we developed a procedure to photochemically convert (6-4) photoproducts to single-strand breaks in UV-irradiated DNA with a background low enough to permit repair measurements. Analysis of a specific DNA sequence indicated that photoinduced alkali-labile sites (PALS) were induced with the same site-specificity as (6-4) photoproducts. Normal human and xeroderma pigmentosum (XP) variant cells rapidly excised (6-4) photoproducts measured as PALS, but little repair was seen in cells from XP complementation group A. These repair kinetics corresponded to those determined in the same samples by radioimmunoassay of (6-4) photoproducts. Thus we conclude that the rapid repair of (6-4) photoproducts observed in UV-irradiated human cells is not the result of a conformational change resulting in epitope loss, but reflects excision of this photoproduct from DNA.     

Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.     

UV mutagenesis, cytotoxicity and split-dose recovery in a human-CHO cell hybrid having intermediate (6-4) photoproduct repair

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCC1, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na+/K+-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5-6) cyclobutane dimers and (6-4) photoproducts was examined by radioimmunoassay; (6-4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCC1 gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6-4) UV photoproduct repair and UV split-dose recovery.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Relationship between pyrimidine dimers, 6-4 photoproducts, repair synthesis and cell survival: studies using cells from patients with trichothiodystrophy

Trichothiodystrophy is a genetic disease which in the majority of cases studied is associated with a deficiency in the ability to repair UV damage in cellular DNA. Three categories of UV response have been identified. In type 1 the response is completely normal, whereas type 2 cells are deficient in excision-repair, with properties indistinguishable from those of XP complementation group D. Type 3 cells have normal survival following UV-irradiation and normal rates of removal of cyclobutane pyrimidine dimer sites. Nevertheless repair synthesis is reduced by 50% in these cell strains and this is associated with a marked reduction in the repair of 6-4 photoproducts from cellular DNA. The present results show that 50% or more of repair synthesis at early times after irradiation of normal primary human fibroblasts is attributable to repair of 6-4 products. They also suggest that repair of cyclobutane dimers is crucial for cell survival.     

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.     

The induction and repair of (6-4) photoproducts in Neurospora crassa.

The (6-4) photoproduct lesion found in DNA after UV irradiation is repaired by germinating Neurospora crassa conidia. Wild-type Neurospora removes 80% of the (6-4) photoproduct in approximately 20 min and maximal repair is accomplished by 30 min with approximately 89% of the original lesions removed. Mutagen-sensitive Neurospora mutants belonging to the established excision repair epistasis group, UVS-2, are not defective in the removal of cyclobutane pyrimidine dimers. Furthermore, we find these mutants capable of removing (6-4) photoproducts from their DNA at a rate similar to wild type. Comparable kinetics are also observed in key members of the other two epistasis groups.     

Xeroderma pigmentosum variant cells are not defective in the repair of (6-4) photoproducts

Using radioimmunoassays specific for (6-4) photoproducts and cyclobutane dimers, Xeroderma pigmentosum variant cells appear to have a normal capacity for the repair of each of these lesions. However, these assays measure an early stage in the repair pathway and we do not exclude the possibility that repair is not successfully completed following UV irradiation and excision of DNA photoproducts.     

(6-4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers

A polyclonal antiserum raised against UV-irradiated DNA can be used to assay cyclobutane pyrimidine dimers and Pyr(6-4)Pyo photoproducts specifically by changing the nature of the 32P-labelled antigen. Pyr(6-4)Pyo photoproducts were removed faster than cyclobutane dimers in UV-irradiated human, hamster and mouse cells. Xeroderma pigmentosum cells from complementation groups A, C and D were deficient in the repair of both lesions.     

Repair of apurinic/apyrimidinic sites by UV damage endonuclease; a repair protein for UV and oxidative damage.

UV damage endonuclease (UVDE) initiates a novel form of excision repair by introducing a nick imme-diately 5" to UV-induced cyclobutane pyrimidine dimers or 6-4 photoproducts. Here, we report that apurinic/apyrimidinic (AP) sites are also nicked by Neurospora crassa and Schizosaccharomyces pombe UVDE. UVDE introduces a nick immediately 5" to the AP site leaving a 3"-OH and a 5"-phosphate AP. Apyrimidinic sites are more effectively nicked by UVDE than apurinic sites. UVDE also possesses 3"-repair activities for AP sites nicked by AP lyase and for 3"-phosphoglycolate produced by bleomycin. The Uvde gene introduced into Escherichia coli cells lacking two types of AP endonuclease, Exo III and Endo IV, gave the host cells resistance to methylmethane sulfonate and t-butyl hydroperoxide. We identified two AP endonuclease activities in S.pombe cell extracts. Besides cyclobutane pyrimidine dimers and 6-4 photoproducts, N. crassa UVDE also nicks Dewar photoproducts. Thus, UVDE is able to repair both of the major forms of DNA damage in living organisms: UV-induced DNA lesions and AP sites.     

The DNA of UV-irradiated normal and excision-deficient mammalian cells undergoes relaxation in an initial stage of DNA repair

Using a radioimmunoassay specific for Pyr(6-4)Pyo photoproducts, we have demonstrated the removal of these lesions from denaturated DNA isolated from UV-irradiated Chinese hamster ovary cells at various times post irradiation. When assayed undenatured, these same DNA samples, which are initially 10-20 times less capable of binding antibody, show a substantial increase in binding capacity during the first few hours of repair. At 3 h post irradiation the difference between native and heat-denatured DNA samples is negligible, indicating that all of the residual lesions are contained in a single-stranded (relaxed) configuration. This relaxation also occurs in UV-hypersensitive cell lines, that are deficient in the ability to remove Pyr(6-4)Pyo photoproducts. Novobiocin, an inhibitor of topoisomerase II, prevents both the initial increase in binding and the subsequent excision of the antibody-binding sites.     

T4 DNA polymerase (3''-5'') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example.

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.     

The induction and repair of lesions produced by the photolysis of (6-4) photoproducts in normal and UV-hypersensitive human cells

A radioimmunoassay was used to study the induction and repair of damage produced by the photolysis of (6-4) photoproducts in normal and UV-sensitive human cells. Photochemical conditions were established to optimize the production of photolyzed (6-4) photoproducts in human cell DNA with minimal induction of other photoproducts. The repair of this photoproduct, presumed to be a Dewar pyrimidinone, was similar to that determined for the (6-4) photoproduct, with most of the antibody-binding sites removed within 4 h post-photolysis. Whereas xeroderma pigmentosum group A cells were deficient in the repair of this lesion, an XP variant and two cell lines selectively hypersensitive to UVB-irradiation were shown to have normal repair. The radioimmunoassay was further used to demonstrate the alkali-lability of the (6-4) photolysis product.     

Intermediate (6-4) photoproduct repair in Chinese hamster V79 mutant V-H1 correlates with intermediate levels of DNA incision and repair replication

A DNA-repair mutant isolated from Chinese hamster V79 cells, V-H1, has been characterized as having only slightly reduced unscheduled DNA synthesis (UDS) and intermediate levels of DNA incision and repair replication after UV exposure. This observation was unexpected, since V-H1 has been shown by genetic complementation analysis to belong to the UV5 complementation class (i.e., class 2), exhibiting equivalent UV hypersensitivity and hypermutability as UV5 cells, which are defective in incision, UDS and repair replication. We have examined the repair of cyclobutane dimers and (6-4) photoproducts in V-H1 and V79 cells and shown that V-H1 cells are deficient in cyclobutane dimer repair, but exhibit intermediate (6-4) photoproduct repair, unlike UV5 cells which are completely deficient in (6-4) photoproduct repair. Our results confirm observations made in other UV-hypersensitive Chinese hamster cell mutants in CHO complementation class 2, and suggest that the gene affected in these mutants (ERCC2) may be involved in at least two distinct repair pathways in hamster cells.     

Retarded excision of pyrimidine dimers in human unstimulated lymphocytes

Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.     

Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.