An immunoassay for measuring repair of UV photoproducts.

A method is described that makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UVC)-pyrimidine-pyrimidone 6-4 photoproducts ([6-4]PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irradiated cells is applied to a nitrocellulose dot-blot and quantitated using an enzyme-conjugated secondary antibody and a color assay. Although the polyclonal antiserum contains antibodies to both [6-4]PPs and CPDs, repair of these lesions can be measured separately by differential destruction or repair of one or other photoproduct. The method is useful for measuring repair in total genomic DNA, and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m(2) of UVC and less. The method is very versatile and has been used to measure repair in human cells, yeasts, plants, archaea, bacteria, and filamentous fungi.

Transcriptional responses to biologically relevant doses of UV-B radiation in the model archaeon, Halobacterium sp. NRC-1.

BACKGROUND: Most studies of the transcriptional response to UV radiation in living cells have used UV doses that are much higher than those encountered in the natural environment, and most focus on short-wave UV (UV-C) at 254 nm, a wavelength that never reaches the Earth's surface. We have studied the transcriptional response of the sunlight-tolerant model archaeon, Halobacterium sp. NRC-1, to low doses of mid-wave UV (UV-B) to assess its response to UV radiation that is likely to be more biologically relevant. RESULTS: Halobacterium NRC-1 cells were irradiated with UV-B at doses equivalent to 30 J/m2 and 5 J/m2 of UV-C. Transcriptional profiling showed that only 11 genes were up-regulated 1.5-fold or more by both UV-B doses. The most strongly up-regulated gene was radA1 (vng2473), the archaeal homologue of RAD51/recA recombinase. The others included arj1 (vng779) (recJ-like exonuclease), top6A (vng884) and top6B (vng885) (coding for Topoisomerase VI subunits), and nrdJ (vng1644) (which encodes a subunit of ribonucleotide reductase). We have found that four of the consistently UV-B up-regulated genes, radA1 (vng2473), vng17, top6B (vng885) and vng280, share a common 11-base pair motif in their promoter region, TTTCACTTTCA. Similar sequences were found in radA promoters in other halophilic archaea, as well as in the radA promoter of Methanospirillum hungatei. We analysed the transcriptional response of a repair-deficient DeltauvrA (vng2636) DeltauvrC (vng2381) double-deletion mutant and found common themes between it and the response in repair proficient cells. CONCLUSION: Our results show a core set of genes is consistently up-regulated after exposure to UV-B light at low, biologically relevant doses. Eleven genes were up-regulated, in wild-type cells, after two UV-B doses (comparable to UV-C doses of 30 J/m2 and 5 J/m2), and only four genes were up-regulated by all doses of UV-B and UV-C that we have used in this work and previously. These results suggest that high doses of UV-C radiation do not necessarily provide a good model for the natural response to environmental UV. We have found an 11-base pair motif upstream of the TATA box in four of the UV-B up-regulated genes and suggest that this motif is the binding site for a transcriptional regulator involved in their response to UV damage in this model archaeon.

The uvrA, uvrB and uvrC genes are required for repair of ultraviolet light induced DNA photoproducts in Halobacterium sp. NRC-1.

BACKGROUND: Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants. RESULTS: Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6-4 photoproducts from their DNA after irradiation with 150 J/m2 of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry. CONCLUSION: Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6-4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.

A dot-blot immunoassay for measuring repair of ultraviolet photoproducts.

The method described here makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UV-C)--pyrimidine-pyrimidone 6-4 photoproducts ([6-4]PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irradiated cells is applied to a nitrocellulose dot-blot and quantified using an enzyme-conjugated secondary antibody and a color assay. Though the polyclonal antiserum contains antibodies to both cyclobutane pyrimidine dimers and (64) photoproducts, repair of these can be measured separately by differential destruction of one or other photoproduct. The method is useful for measuring repair in total genomic DNA. It is more sensitive than most other methods and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m2 of UV-C in DNA from mammalian cells.

UV irradiation induces homologous recombination genes in the model archaeon, Halobacterium sp. NRC-1.

BACKGROUND: A variety of strategies for survival of UV irradiation are used by cells, ranging from repair of UV-damaged DNA, cell cycle arrest, tolerance of unrepaired UV photoproducts, and shielding from UV light. Some of these responses involve UV-inducible genes, including the SOS response in bacteria and an array of genes in eukaryotes. To address the mechanisms used in the third branch of life, we have studied the model archaeon, Halobacterium sp. strain NRC-1, which tolerates high levels of solar radiation in its natural hypersaline environment. RESULTS: Cells were irradiated with 30-70 J/m(2) UV-C and an immunoassay showed that the resulting DNA damage was largely repaired within 3 hours in the dark. Under such conditions, transcriptional profiling showed the most strongly up-regulated gene was radA1, the archaeal homolog of rad51/recA, which was induced 7-fold. Additional genes involved in homologous recombination, such as arj1 (recJ-like exonuclease), dbp (eukaryote-like DNA binding protein of the superfamily I DNA and RNA helicases), and rfa3 (replication protein A complex), as well as nrdJ, encoding for cobalamin-dependent ribonucleotide reductase involved in DNA metabolism, was also significantly induced in one or more of our experimental conditions. Neither prokaryotic nor eukaryotic excision repair gene homologs were induced and there was no evidence of an SOS-like response. CONCLUSION: These results show that homologous recombination plays an important role in the cellular response of Halobacterium sp. NRC-1 to UV damage. Homologous recombination may permit rescue of stalled replication forks, and/or facilitate recombinational repair. In either case, this provides a mechanism for the observed high-frequency recombination among natural populations of halophilic archaea.

An immunoassay for measuring repair of ultraviolet photoproducts.

A method is described that makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UVC)-pyrimidine-pyrimidone 6-4 photoproducts ([6-4]PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irradiated cells is applied to a nitrocellulose dot blot and quantitated using an enzyme-conjugated secondary antibody and a color assay. Although the polyclonal antiserum contains antibodies to both [6-4]PPs and CPDs, repair of these can be measured separately by differential destruction or repair of one or other photoproduct. The method is useful for measuring repair in total genomic DNA and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m2 of UVC and less. The method is very versatile and has been used to measure repair in human cells, yeasts, plants, archaea, bacteria, and filamentous fungi.