Rapid immunoassays for detection of UV-induced cyclobutane pyrimidine dimers in whole bacterial cells

Immunoassays were developed to measure DNA damage retained by UV-irradiated whole bacterial cells. Active Mycobacterium parafortuitum and Serratia marcescens cells were fixed and incubated with cyclobutane pyrimidine dimer-binding antibodies after being exposed to known UV doses (254 nm). When both fluorescent (Alexa Fluor 488) and radiolabeled ((125)I) secondary antibodies were used as reporters, indirect whole-cell assays were sensitive enough to measure intracellular UV photoproducts in M. parafortuitum and S. marcescens cells as well as photoenzymatic repair responses in S. marcescens cells. For the same UV dose, fluorescent DNA photoproduct detection limits in whole-cell assays (immunofluorescent microscopy) were similar to those in fluorescent assays performed on membrane-bound DNA extracts (immunoslot blot). With either fluorescent or radiolabeled reporters, the intracellular cyclobutane pyrimidine dimer content of UV-irradiated whole bacterial cells could be reliably quantified after undergoing a <0.5-order-of-magnitude decrease in culturability. Immunofluorescent microscopy results showed that photoenzymatic repair competence is not uniformly distributed among exponential-growth UV-irradiated pure cultures.     

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.     

Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice

During evolution, placental mammals appear to have lost cyclobutane pyrimidine dimer (CPD) photolyase, an enzyme that efficiently removes UV-induced CPDs from DNA in a light-dependent manner. As a consequence, they have to rely solely on the more complex, and for this lesion less efficient, nucleotide excision repair pathway. To assess the contribution of poor repair of CPDs to various biological effects of UV, we generated mice expressing a marsupial CPD photolyase transgene. Expression from the ubiquitous beta-actin promoter allowed rapid repair of CPDs in epidermis and dermis. UV-exposed cultured dermal fibroblasts from these mice displayed superior survival when treated with photoreactivating light. Moreover, photoreactivation of CPDs in intact skin dramatically reduced acute UV effects like erythema (sunburn), hyperplasia and apoptosis. Mice expressing the photolyase from keratin 14 promoter photo reactivate CPDs in basal and early differentiating keratinocytes only. Strikingly, in these animals, the anti-apoptotic effect appears to extend to other skin compartments, suggesting the presence of intercellular apoptotic signals. Thus, providing mice with CPD photolyase significantly improves repair and uncovers the biological effects of CPD lesions.     

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.     

Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells

The time courses of excision repair and photoreactivation of pyrimidine dimers induced by 254-nm UV were examined in the genome overall and in the c-ras sequence of RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Excision repair was more efficient in the ras sequence than in the genome overall, whereas no differences in efficiency of photoreactivation were detected. These results suggest that excision repair is affected by the accessibility of chromatin, while photoreactivation is not.     

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K⁺ solution, whereas a potentially photoreactive basket conformation is favored in Na⁺ solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na⁺ solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K⁺ solution. In the current study, Na⁺ was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K⁺ solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K⁺ solution are dynamic and able to access photoreactive conformations more easily than in Na⁺ solution.     

DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers

Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only approximately 25% of mutations occur at thymine (T), whilst 75% are targeted to cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of poleta, where approximately 45% of mutations occur at Ts and approximately 55% at Cs. At the present time, it is unclear whether the C-->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to uracil (U). In order to assess the role that human poliota might play, if any, in the replicative bypass of such UV-photoproducts, we have analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurs more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only two-fold less frequently than the correct Watson-Crick base, A. While pol iota generally extended the G:3'U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to, or greater than that observed with human pols eta and kappa and S. cerevisiae pol zeta under the same assay conditions. Thus, we hypothesize that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.     

Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers.

The effects of short wave ultraviolet (UV)-induced DNA lesions on the catalytic activity of Drosophila melanogaster topoisomerase II were investigated. The presence of these photoproducts impaired the enzyme's ability to relax negatively supercoiled pBR322 plasmid molecules. As determined by DNA photolyase-catalyzed photoreactivation experiments, enzyme inhibition was due to the presence of cyclobutane pyrimidine dimers in the DNA. When 10-20 cyclobutane dimers were present per plasmid, the initial velocity of topoisomerase II-catalyzed DNA relaxation was inhibited approximately 50%. Decreased relaxation activity correlated with an inhibition of the DNA strand passage step of the enzyme's catalytic cycle. In contrast, UV-induced photoproducts did not alter the prestrand passage DNA cleavage/religation equilibrium of topoisomerase II either in the absence or presence of antineoplastic agents. Results of the present study demonstrate that the repair of cyclobutane pyrimidine dimers is important for the efficient catalytic function of topoisomerase II.     

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.     

Proximity of repair patches to persistent pyrimidine dimers in DNA of normal human and xeroderma pigmentosum cells

The proximity of repair patches to persistent pyrimidine dimers in normal human cells and xeroderma pigmentosum group C and D cells was analyzed by sequential digestion of repaired DNA with Micrococcus luteus UV-endonuclease and Escherichia coli DNA polymerase I. Although this enzymatic digestion removed one-third of the pyrimidine dimers, less than 3% of the label associated with repair patches and a similar amount of uniformly labeled DNA were removed. The repair patches therefore appear to be similarly distant from persistent dimers in all cell types, and, in particular, are not adjacent to unexcised dimers in xeroderma pigmentosum group D cells. A previous model that suggested that patches are inserted adjacent to dimers in xeroderma pigmentosum group D cells receives no support from these results.