Genetic and Molecular Characteristics of X-Ray-Sensitive Mutants of Escherichia coli Defective in Repair Synthesis

Alleles responsible for X-ray-sensitive characteristics of three mutants of Escherichia coli B, which were also sensitive to ultraviolet (UV) irradiation, were mapped near metE locus, and named res-1, res-2, and res-3. All the res(-) mutants showed no host cell reactivability (Hcr(-)) for transducing deoxyribonucleic acid (DNA) of P1 phage irradiated by UV but they were Hcr(+) for infective DNA of P1 phage. Furthermore, they showed no detectable activity of DNA polymerase. Characteristics of allele res-1 were studied in detail. The mutant res-1 uvr(+) showed an extensive degradation of DNA after UV irradiation. Double mutants carrying res-1 uvrA(-), res-1 uvrB(-), and res-1 uvrC(-) showed no marked increase in UV sensitivity beyond that of the uvr(-) single mutants and only negligible UV-induced DNA degradation. The uvr(-) mutations showed no such suppressive effect on DNA degradation induced by X rays in these double mutants. It is concluded that res(-) mutants are defective in the second step (repair synthesis) of the excision repair process and that DNA polymerase is partly responsible for the assumed resynthesis step.     

Study of MFD in Bacillus subtilis.

The frequency of UV-induced extragenic suppressor reversions to leucine independence in B. subtilis carrying a leu8 mutation decreased when irradiated cells were temporarily incubated in medium deprived of nitrogen sources. This mutation frequency decline (MFD) was inhibited by acriflavine and was poorly expressed in a uvr1 mutant. Consequently, MFD may be considered as the manifestation of an anti-mutagenic activity of excision repair. MFD was decelerated and even vanished in cells subjected to prolonged starvation of nitrogen sources before irradiation. MFD was accelerated in bacteria that were first irradiated and incubated in nutritional medium for at least 30 min. The stimulation of MFD by UV exposure was observed only in the uvr+ strain and depended on protein synthesis after irradiation. It is assumed that different rates of MFD in cells of various pre-radiation histories reflect different levels of the excision-repair activity inherent in these cells.     

Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1

To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3' splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.     

Mutagen sensitivity of Drosophila melanogaster. VI. Evidence from the excision-defective mei-9AT1 mutant for the timing of DNA-repair activity during spermatogenesis

The sex-linked recessive lethal test has been used to compare mutation induction by ethyl methanesulfonate and methyl methanesulfonate in spermatogenic stages of the DNA repair-deficient mei-9AT1 mutant and a repair-proficient control strain. For both agents, the data demonstrate that induced mutation rates are similar in both strains for the meiotic and post-meiotic broods. Conversely, for spermatogonial broods, the data indicate that the excision-deficient strain exhibits a 4-8 fold increase in induced mutation rate in comparison to the excision-proficient control strain. These experiments suggest that the low mutability of gonial cells normally observed for these agents is due to effective excision-repair processes which function until the commencement of meiosis. From alkylation mutagenesis experiments with repair-deficient E. coli strains, we note that the mei-9 strain exhibits pleiotropic mutant phenotypes very similar to those displayed by the uvr D mutant. By analogy with these studies, we speculate that mei-9, like uvr D, is deficient in a DNA unwinding protein.     

Ultraviolet B radiation enhances a phytochrome-B-mediated photomorphogenic response in Arabidopsis

Ultraviolet B radiation (UV-B, 290-315 nm) can cause damage and induce photomorphogenic responses in plants. The mechanisms that mediate the photomorphogenic effects of UV-B are unclear. In etiolated Arabidopsis seedlings, a daily exposure to 2.5 h of UV-B enhanced the cotyledon opening response induced by a subsequent red light (R) pulse. An R pulse alone, 2.5 h of UV-B terminated with a far-red pulse, or 2.5 h of continuous R caused very little cotyledon opening. The enhancing effect of UV-B increased with fluence rate up to approximately 7.58 micromol m(-2) s(-1); at higher fluence rates the response to UV-B was greatly reduced. The phyA, phyA cry1, and cry1 cry2 mutants behaved like the wild type when exposed to UV-B followed by an R pulse. In contrast, phyB, phyB cry1, and phyB phyA mutants failed to open the cotyledons. Thus, phytochrome B was required for the cotyledon opening response to UV-B --> R treatments, whereas phytochrome A and cryptochromes 1 and 2 were not necessary under the conditions of our experiments. The enhancing effect of low doses of UV-B on cotyledon opening in uvr1 uvr2 and uvr1 uvr3 mutants, deficient in DNA repair, was similar to that found in the wild type, suggesting that this effect of UV-B was not elicited by signals derived from UV-B-induced DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts). We conclude that low doses of UV-B, perceived by a receptor system different from phytochromes, cryptochromes, or DNA, enhance a de-etiolation response that is induced by active phytochrome B.     

Photochemistry and photobiology of 5-azacytidine: effect of repair on photostabilization of Escherichia coli.

The photochemical stability of the anomalous nucleic acid base 5-azacytidine (z5Cyd) on irradiation at 254 nm is by about one order of magnitude less than that of cytidine (Cyd). Contrary to the photochemical behaviour, incorporation of z5Cyd into the nucleic acids of E. coli strains SR 20 (uvr+ rec+), SR 74 (uvr+ rec-) and SR 22 (uvr- rec+) produced a higher resistance to UV light. Only the SR 73 (uvr- rec-) strain was shown to have an increased UV sensitivity. This latter finding is in accord with the photochemical properties of z5Cyd. The results led to the conclusion that excision and recombination repair processes contribute to the observable protective effect. The fact that inhibition of excission repair by caffeine or proflavine of the mutant uvr+ rec- changes protection into sensitization supports this idea.     

Ultraviolet Mutagenesis and Its Repair in an ESCHERICHIA COLI Strain Containing a Nonsense Codon

Ultraviolet mutagenesis and its repair were studied mainly in WU36-10-89, a uvr(-) strain of Escherichia coli containing a UAG mutation in a gene for leucine biosynthesis. Following ultraviolet (UV) irradiation revertants appearing with or without direct photoreactivation (PR) were classified according to the presence and type of suppressor they contained. We find UV mutation production to be quite specific. An analysis of revertants produced by UV indicates they are formed mainly from GC --> AT and that the miscoding is due to a cytosine residue at the site of mutation in a cytosine-thymine (CT) dimer. We propose that the dimer serves as template during some aspects of repair replication and at the time of replication the C in the dimer directs the insertion of A in the complementary strand. We also note that C --> A and T -->G changes caused by a CT dimer occur much less frequently.     

Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

Induction by UV light of the SOS function sfiA in Escherichia coli strains deficient or proficient in excision repair.

The influence of the nucleotide excision repair system on the induction by UV irradiation of the SOS function sfiA has been investigated. The level of sfiA expression was monitored by means of a sfiA::lacZ operon fusion in both the wild-type strain and a uvrA mutant. We found that the initial steady rate of sfiA expression was proportional to the UV dose and was identical in uvr+ and uvrA backgrounds. This suggests that the initial steady rate of sfiA expression is determined by the initial number of lesions and before any effect of excision repair. We confirmed that after 2 h of expression the net synthesis of sfiA product is, for the same UV dose, about five times lower in uvr+ than in uvrA strains. We show that this is due to earlier repression of the SOS system in uvr+ than in uvrA strains and not to different initial rates.     

Mutants of Yeast Sensitive to Ultraviolet Light

Six uvr mutants of Saccharomyces cerevisiae with hypersensitivity to ultraviolet (UV) light were isolated after mutagen treatment with ethylmethanesulfonate. UV sensitivity ranges from moderate to extreme, and four of the mutants are also sensitive to nitrous acid. Ranking in terms of UV sensitivity does not parallel ranking in terms of nitrous acid sensitivity. Homozygous diploid mutant strains are somewhat less sensitive than the corresponding haploids. All mutations are recessive. None of the mutants is sensitive to gamma rays, and each shows photoreactivation after UV radiation. Complementation tests and tetrad analysis indicate that each strain represents mutation in a different gene. Two of the uvr genes are linked, and two others are centromere-linked.     

Ultraviolet-B radiation impacts light-mediated turnover of the photosystem II reaction center heterodimer in Arabidopsis mutants altered in phenolic metabolism

Ultraviolet-B (UV-B) radiation can have a negative impact on the growth and development of plants. Plants tolerant to UV-B alleviate these effects using UV-screening pigments that reduce the penetration of UV-B into mesophyll tissue. Little is known about the relative contribution of specific phenolic compounds to the screening capacity of leaves. The D1 and D2 proteins constituting the photosystem (PS) II reaction center heterodimer are targets of UV-B radiation and can be used as an in situ sensor for UV penetration into photosynthetic tissue. Degradation of these proteins occurs under very low fluences of UV-B, and is strongly accelerated in the presence of visible light. Using the D1-D2 degradation assay, we characterized UV-B sensitivity of Arabidopsis mutants (tt4, tt5, and fah1) that are genetically altered in their composition of phenolic compounds. We found that changes in phenol metabolism result in altered rates of PSII reaction center heterodimer degradation under mixtures of photosynthetically active radiation and UV-B. A comparison of D2 degradation kinetics showed increased UV sensitivity of the Landsberg (Landsberg erecta) tt5 mutant relative to the Landsberg tt4 mutant and the Landsberg wild type. Despite a lack of flavonoid accumulation, the tt4 mutant is not particularly UV sensitive. However, the tolerance of this mutant to UV-B may reflect the increased accumulation of sinapate esters that strongly absorb in the UV range, and may thus protect the plant against environmentally relevant UV-B radiation. This sinapate-mediated protection is less obvious for the tt4 mutant of Columbia ecotype, indicating that the relative contribution of particular phenolics to the total screening capacity varies with the genetic background. The role of sinapate esters in UV screening is further substantiated by the results with the fah1 mutant where absence of most of the sinapate esters results in a significantly accelerated degradation of D2 under mixed light conditions. Because the latter mutant is not expected to be deficient in flavonoids, the relative contribution of flavonoids as protectants of PSII reaction center heterodimer against UV-B damage in Arabidopsis needs to be re-evaluated vis-a-vis screening by simple phenolics like sinapate esters.     

光受体UVR8对UV-B响应机制研究进展

来自太阳光谱中的UV-B辐射被认为是一种重要的环境信号,可以被植物感受并诱导植物调整自身生长和发育状态以适应环境。人们对植物中光敏色素、隐花色素和蓝光受体向光素的研究已非常深入,但对植物响应UV-B的机制仅在最近才取得一些突破性进展。这些研究发现,植物中存在着UV-B受体UVR8(UV Resistance Locus 8)。目前认为,UVR8二聚体感应UV-B后瞬间解聚为单体,并与E3泛素连接酶COP1(constitutively photomorphogenic 1)相互作用,从而激活UV-B响应基因的表达。该文从UVR8的发现、UVR8的结构和感受UV-B机制、UVR8二聚体重新形成以及UV-B信号传导与可见光信号传导途径间的差异等方面综述了关于UV-B受体UVR8的最新研究成果。     

Specifically UV-sensitive photoreactivable mutant of Salmonella abony

A new type of UV-sensitive mutant was isolated in Salmonella abony. The war 12 mutation causing UV sensitivity did not affect photoreactivability of UV damage or sensitivity to γ-rays, methyl methanesulfonate (MMS), mitomycin (MC) or 4-nitro-quinoline I-oxide (4NQO).Mutation uvr I2 appeared to be near the uvr B gene of Salmonella: the frequencies of contransduction of uvr B2 and uvr I2 mutations with gal were found to be 3% and 6% respectively.Close localization of the uvr I2 and uvr B2 mutations, the possibility of recombination between them and their phenotypic differences (both uvr B2 and uvr I2 mutants show quantitatively different Hcr phenotypes and different sensitivities to MC and 4NQO) suggest that the uvr B2 and uvr I2 mutations might be localized in different cistrons of an operon controlling the first step of excision repair.     

Severe sensitivity to ultraviolet radiation in anArabidopsis mutant deficient in flavonoid accumulation

A mutantArabidopsis thaliana L., which displays a dramatic increase in sensitivity to ultraviolet-B (UV-B) radiation compared with wild-type plants, has been isolated by chemical mutagenesis. This mutation appears to affect UV-tolerance specifically, since mutant plants are indistinguishable from wild type with respect to their degree of resistance to other forms of stress. The UV-sensitive mutation proved to be recessive and to segregate as a single Mendelian locus. This single gene defect was shown to lead to a block in the synthesis of a group of flavonoids which normally accumulate in developing wild-typeArabidopsis and which increase in concentration when plants are exposed to UV radiation. One of these compounds has been identified as a rhamnosylated derivative of the flavonol, kaempferol. The results suggest that one or more of the flavonoids whose production has been blocked in the UV-sensitive mutant is essential for the protection ofArabidopsis against UV-radiation damage. This constitutes further evidence that flavonoids play an important role in the protection of plants from the damaging effects of UV-B light.Abbreviations EMS ethyl methyl sulfonate - NMR nuclear magnetic resonance - uvs UV-sensitive Dedicated to Professor Hartmut K. Lichtenthaler on the occasion of his 60th birthdayThis work was supported by Cooperative State Research Service, U.S. Department of Agriculture, under Agreements Nos. 90-37280-5664 and 90-372780-5808 and by National Science Foundation Grant MCB 93-16496. We wish to thank Lola Peñarrubia, Elena del Campillo, Patrick Neil and Julie Montgomery for innumerable and fruitful discussions.     

Suppression of Ultraviolet Sensitivity in Escherichia coli uvr502 by the su58 Missense Suppressor

Introduction of the su58 missense suppressor into the chromosome of the uvr502 mutant, either by mutation or by transduction, results in a marked increase of ultraviolet resistance of the uvr502 mutant. In the uvr(+) genetic background, the su58 suppressor causes some decrease of ultraviolet resistance and marked increase in the spontaneous mutation frequency. The presence of the su58 suppressor did not decrease the high frequency of spontaneous mutants in the population of the uvr502 strain. However, the significant increase in spontaneous mutant frequency in the uvr(+)su58 strain makes the difference between the uvr502 su58 and the uvr(+)su58 strains 18 times lower than that between the uvr502 and the uvr(+) suppressor-free strains. Since the missense suppressors act at the level of translation, the results suggest that the product of the uvr(502) gene is a protein.     

Analysis of repair processes by the determination of the induction of cell killing and mutations in two repair-deficient Chinese hamster ovary cell lines

Two UV sensitive DNA-repair-deficient mutants of Chinese hamster ovary cells (43-3B and 27-1) have been characterized. The sensitivity of these mutants to a broad spectrum of DNA-damaging agents: UV254nm, 4-nitroquinoline-1-oxide (4NQO), X-rays, bleomycin, ethylnitrosourea (ENU), ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS) and mitomycin C (MMC) has been determined. Both mutants were not sensitive to X-rays and bleomycin. 43-3B was found to be sensitive to 4NQO, MMC and slightly sensitive to alkylating agents. 27-1 was sensitive only to alkylating agents. The results suggest the existence of two repair pathways for UV-induced cytotoxicity: one pathway which is also used for the removal of 4NQO and MMC adducts and a second pathway which is also used for the removal of alkyl adducts. Parallel to the toxicity, the induction of mutations at the HPRT and Na+/K+-ATPase loci was determined. The increased cytotoxicity to UV, MMC and 4NQO in 43-3B cells and the increased cytotoxicity to UV in 27-1 cells correlated with increased mutability. It was observed that the increase in mutation induction at the HPRT locus was higher than that at the Na+/K+-ATPase locus. As only point mutations give rise to viable mutants at the Na+/K+-ATPase locus the lower mutability at this locus suggests that defective excision repair increases the chance for deletions. Despite an increased cytotoxicity to ENU in 27-1 cells the mutation induction by ENU was the same in 27-1 and wild-type cells at both loci, which suggests that the mutations are mainly induced by directly miscoding adducts (e.g. O-6 alkylguanine), which cannot be removed by CHO cells. As EMS and MMS treatment of 27-1 cells caused an increase in mutation induction at the HPRT locus and a decrease at the Na+/K+-ATPase locus it indicates that these agents induce a substantial fraction of other mutagenic lesions, which can be repaired by wild-type cells. This suggests that O-6 alkylation is not the only mutagenic lesion after treatment with alkylating agents.