Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis.

Summary The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage. In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied. Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light-independent repair. Four of these were located in amino acid residues between Gln333 and Leu371 near the 3 end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50. Three mutants that had insertions located in the 42 by segment from 399 to 441 by of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation. These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light-independent repair is at least in part distinct from that necessary for light-dependent photoreactivation.     

Effect of photoreactivating light on survival of ultraviolet-light-irradiatedStreptomyces lividans 66

Summary Biological systems can repair damage induced in their DNA by ultraviolet light (UV). Most cells contain at least three DNA repair pathways, each of which has a marked effect on UV survival. Excision repair and recombinational (postreplication) repair are light-independent whereas photoreactivation (PR), whether enzyzmatic or photochemical, is light-dependent. The specificity of photoreactivation for UV-induced DNA damage allows it to be used as a tool for examining whether premutational DNA lesions are preferred sites for photoreversal; it therefore plays an important role in mutagenesis studies. Evidence is presented here that PR occurs in a time-dependent fashion in three strains ofStreptomyces lividans 66. The effect appears to be independent of temperature and is observed only when PR treatment is given after UV irradiation. The present experiments do not discriminate between enzymatic and photochemical protection.     

Photoreactivation of UV-irradiated blue-green algae and algal virus LPP-1

Ultraviolet (UV) sensitivity and photoreactivation of blue-green algae Cylindrospermum sp., Plectonema boryanum, spores of Fischerella muscicola and algal virus (cyanophage) LPP-1 were studied. The survival value after UV irradiation of filaments of Cylindrospermum sp. and Virus LPP-1 showed exponential trend and these were comparatively sensitive towards UV than F. muscicola and P. boryanum. Photoreactivation of UV-induced damage occurred in black, blue, green, yellow, red and white light in Cylindrospermum sp., however only black, blue and white light were capable of photorepair of UV-induced damage in P. boryanum, spores of F. muscicola and virus LPP-1 in infected host alga. Pre-exposure to yellow and black light did not show photoprotection. The non-heterocystous and nitrogen fixation-less mutants of Cylindrospermum sp. were not induced by UV and their spontaneous mutation frequency was not affected after photoreactivation. The short trichome mutants of P.boryanum were more resistant towards UV.The occurrence of photoreactivation of UV-induced killing in wide range of light in Cylindrospermum sp. is the first report in organisms.     

The Oenothera plastome mutator: effect of UV irradiation and nitroso-methyl urea on mutation frequencies

Summary Oenothera plants homozygous for a recessive plastome mutator allele (pm) showed spontaneous mutation frequencies for plastome genes that are 200-fold higher than spontaneous levels. Mutations occurred at high frequencies in plants grown in the field, in a glass-house, or as leaf tip cultures under fluorescent light, indicating that the plastome mutator activity is UV-independent. However, the chlorotic sectors became visible at an earlier stage of development when seedlings were irradiated, compared to seedlings that were not exposed to UV. These results imply that the rate of sorting-out was increased by the irradiation treatment, possibly due to a decrease in the effective number of multiplication-competent plastids, or a reduction in the extent of cytoplasmic mixing. Nitroso-methyl urea treatment of seeds had a dramatic effect on mutation frequency in both wild-type and plastome mutator samples. When the background mutation rates were low, the combination of the plastome mutator nucleus and the chemical mutagenesis treatment resulted in a synergistic effect, suggesting that the plastome mutator may involve a cpDNA repair pathway.     

Ultraviolet Mutagenesis Studies of [psi], a Cytoplasmic Determinant of SACCHAROMYCES CEREVISIAE

UV mutagenesis was used to probe the molecular nature of [psi], a non-mitochondrial cytoplasmic determinant of Saccharomyces cerevisiae involved in the control of nonsense suppression. The UV-induced mutation from [psi⁺] to [psi^-] showed characteristics of forward nuclear gene mutation in terms of frequency, induction kinetics, occurrence of whole and sectored mutant clones and the effect of the stage in the growth cycle on mutation frequency. The involvement of pyrimidine dimers in the premutational lesion giving the [psi^-] mutation was demonstrated by photoreactivation. UV-induced damage to the [psi] genetic determinant was shown to be repaired by nuclear-coded repair enzymes that are responsible for the repair of nuclear DNA damage. UV-induced damage to mitochondrial DNA appeared to be, at least partly, under the control of different repair processes. The evidence obtained suggests that the [psi] determinant is DNA.     

Liquid holding recovery and photoreactivation of UV-induced damage inStreptococcus lactis

Summary Repair of ultraviolet-light-induced DNA damage inStreptococcus lactis has been examined. The wild-type strain and its derivative Lac^– possess a dark repair system (maximal increase in survival of 4-fold). Enzymatic photoreactivation exists in the two strains but a weaker photoreactivability was found in the Lac^– derivative (4 and 2-fold, respectively). Concomitant reduction of UV-induced mutagenesis (Rif^r marker) was also studied during these two repair phenomena. The absence of dark repair after saturation of photoreactivation suggests that photoreactivation is much more efficient with pyrimidine dimers as substrate.     

Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

UV hyper‐resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase

Exposure to solar radiation can cause mortality in natural communities of pico‐phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV‐hyper‐resistant variant of high light‐adapted strain MED4. The hyper‐resistant strain was constitutively upregulated for expression of the mutT‐phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8‐oxo‐dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error‐free, in contrast to error‐prone SOS dark repair systems that are widespread in bacteria. The UV‐hyper‐resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT‐phrB operon. Two subsequent enrichments for MED4 UV‐hyper‐resistant strains from MED4 wild‐type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper‐resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.     

Molecular cloning and isolation of a cyanobacterial gene which increases the UV and methyl methanesulphonate survival of recA strains of Escherichia coli K12

The unicellular cyanobacterium Gloeocapsa alpicola contains both photoreactivation and excision repair mechanisms for correcting UV-induced damage to its cellular DNA. An 11.5 kb EcoRI fragment was isolated from a cosmid bank of G. alpicola and was shown to complement a recA deletion in Escherichia coli S.17 and JC10289. These recA strains showed increased survival to UV and methyl methanesulphonate (MMS) when transformed with the cyanobacterial DNA fragment, and also showed filamentation in response to UV irradiation. Preliminary analysis of the protein encoded by the cyanobacterial DNA fragment indicated a major protein of 39,000 Da; this is very similar in size to the recA protein of E. coli.     

Radiation Genetics in Microorganisms and Evolutionary Considerations

Recent knowledge of UV-resistance mechanisms in microorganisms is reviewed in perspective, with emphasis on E. coli. Dark-repair genes are classified into "excision" and "tolerance" (ability to produce a normal copy of DNA from damaged DNA). The phenotype of DNA repair is rather common among the microorganisms compared, and yet their molecular mechanisms are not universal. In contrast, DNA photoreactivation is the simplest and the most general among these three repair systems. It is proposed that DNA repair mechanisms evolved in the order: photoreactivation, excision repair, and tolerance repair. The UV protective capacity and light-inducible RNA photoreactivation possessed by some plant viruses are interpreted to be the result of solar UV selection during a rather recent era of evolution.     

UV-B-induced DNA damage and repair pathways in polar Pseudogymnoascus sp. from the Arctic and Antarctic regions and their effects on growth,pigmentation and conidiogenesis

Solar radiation regulates most biological activities on Earth. Prolonged exposure to solar UV radiation can cause deleterious effects by inducing two major types of DNA damage, namely, cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts. These lesions may be repaired by the photoreactivation (Phr) and nucleotide excision repair (NER) pathways; however, the principal UV-induced DNA repair pathway is not known in the fungal genus Pseudogymnoascus. In this study, we demonstrated that an unweighted UV-B dosage of 1.6 kJ m⁻² d⁻¹ significantly reduced fungal growth rates (by between 22% and 35%) and inhibited conidia production in a 10 d exposure. The comparison of two DNA repair conditions, light or dark, which respectively induced photoreactivation (Phr) and NER, showed that the UV-B-induced CPDs were repaired significantly more rapidly in light than in dark conditions. The expression levels of two DNA repair genes, RAD2 and PHR1 (encoding a protein in NER and Phr respectively), demonstrated that NER rather than Phr was primarily activated for repairing UV-B-induced DNA damage in these Pseudogymnoascus strains. In contrast, Phr was inhibited after exposure to UV-B radiation, suggesting that PHR1 may have other functional roles. We present the first study to examine the capability of the Arctic and Antarctic Pseudogymnoascus sp. to perform photoreactivation and/or NER via RT-qPCR approaches, and also clarify the effects of light on UV-B-induced DNA damage repair in vivo by quantifying cyclobutene pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts. Physiological response data, including relative growth rate, pigmentation and conidia production in these Pseudogymnoascus isolates exposed to UV-B radiation are also presented.     

Chloroplast mutations induced by 9-aminoacridine hydrochloride are independent of the<Emphasis Type="Italic"> plastome mutator</Emphasis> in<Emphasis Type="Italic"> Oenothera</Emphasis>

Oenothera plants homozygous for the recessive plastome mutator allele (pm) show chloroplast DNA (cpDNA) mutation frequencies that are about 1,000-fold higher than spontaneous levels. The pm-encoded gene product has been hypothesized to have a function in cpDNA replication, repair and/or mutation avoidance. Previous chemical mutagenesis experiments with the alkylating agent nitroso-methyl urea (NMU) showed a synergistic effect of NMU on the induction of mutations in the pm line, suggesting an interaction between the pm-encoded gene product and one of the repair systems that corrects alkylation damage. The goal of the experiments described here was to examine whether the pm activity extends to the repair of damage caused by non-alkylating mutagens. To this end, the intercalating mutagen, 9-aminoacridine hydrochloride (9AA) was tested for synergism with the plastome mutator. A statistical analysis of the data reported here indicates that the pm-encoded gene product is not involved in the repair of the 9AA-induced mutations. However, the recovery of chlorotic sectors in plants derived from the mutagenized seeds shows that 9AA can act as a mutagen of the chloroplast genome.Communicated by R. Hagemann     

Expression of an Escherichia coli phr gene in the yeast Saccharomyces cerevisiae

Summary A 2 kb DNA fragment, containing the photoreactivation gene phr1 from Escherichia coli, was inserted at the BamH1 site in the tet gene of the yeast — E. coli shuttle vector pJDB207. Photoreactivation — deficient Saccharomyces cerevisiae cells transformed with this plasmid showed photoreactivation of killing after UV irradiation of the cells, while extracts of transformed cells exhibited photoreactivating activity in vitro. Far more photoreactivating enzyme molecules were found when the gene was inserted in the plasmid in the opposite orientation to the tet gene as compared with a plasmid carrying the inserted gene in the same orientation. Photoreactivating enzyme encoded by the E. coli phr1 gene and produced in transformed yeast cells has characteristics of the E. coli photoreactivating enzyme (flavoprotein) as judged from the influence of ionic strength on photoreactivating activity.     

A comparative study of the effects of UV irradiation upon diploid cultures of yeast defective at the rad 3 locus

Summary Therad 3 gene ofSaccharomyces cerevisiae appears to code for one of the enzymes involved in the repair of UV induced pyrimidine dimers. Haploid and diploid yeast cultures carrying different mutant alleles of therad 3 gene show considerable variation in their responses to both UV inactivation and post UV modifying treatments such as liquid holding in basal medium and photoreactivation. Positive liquid holding recovery was shown only by those diploid cultures containing alleles which conferred the highest levels of UV resistance. The results indicate that liquid holding recovery in yeast requires the activity of the excision-repair pathway for expression.     

Cyanobacteria toxins in the Salton Sea

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.     

DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6–4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.     

Oenothera chloroplast DNA polymorphisms associated with plastome mutator activity

Summary Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Chloroplast DNA (cpDNA) was isolated from nine mutants and two green isolates of the plastome mutator line. cpDNA restriction patterns were compared to cpDNA from a representative of the progenitor Johansen strain, and cpDNAs from all eleven plastome mutator lines show changes of fragment mobility due to deletion events at five discrete regions of the plastome. Most of the mutants have cpDNA restriction patterns identical to that of one of the green isolates from the plastome mutator line, and therefore, most of the differences in fragment length are probably not responsible for the mutant phenotypes. In contrast to the plastome mutator line, cpDNA from several populations of a closely related wild-type Oenothera species have few restriction fragment length polymorphisms. This suggests that both mutation frequencies and site-specific cpDNA deletions are elevated in the plastome mutator line, and implicates a defect in the cpDNA repair or replication machinery.     

Comparative studies of chloroplastic and nuclear DNA repair abilities after ultraviolet irradiation of Euglena gracilis

Summary Studies of nuclear and chloroplastic-DNA repair after ultraviolet irradiation of Euglena gracilis show that photoreactivation is very efficient at both the nuclear and chloroplastic level. Liquid-holding or split-dose experiments and treatment with caffeine reveal, furthermore, that dark-repair is very efficient in nuclear DNA but not in chloroplastic DNA (ctDNA). The possibility of a chloroplastic dark-repair of restricted efficiency is discussed.Determination of chloroplastic DNA content by reassociation kinetics indicates that an important degradation follows UV irradiation during liquid holding in the dark.