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.     

DNA repair in competent cells of Bacillus subtilis.

A series of isogenic transformable strains of Bacillus subtilis carrying the uvr-19 or rec-43 mutation or both were constructed. Both mutations made competent cells defective in repairing UV-irradiated cellular or transforming DNA, and their effects were additive in a doubly deficient strain, suggesting that two repair processes, requiring uvr-19+ and rec-43+ gene products, are independently functional in competent cells of B. subtilis.     

Photosensitizing effect of 8-methoxypsoralen on bacteriophage cd

Mutagenesis in extracellular phage sd by 8-metoxypsoralen (8-MOP) and longwave (lambda greater than 310 nm) UV-irradiation has been established. The kinetics of lethal and mutagenic effects of 8-MOP+light was studied. The efficiency of mutagenesis on the first linear part of mutation curve was 0.3% per the lethal hit which is 2 times lower than that of shortwave (lambda=254 nm) UV-irradiation. The maximum yield of mutants makes up 1%, after which the mutation curve is maintained. It has been established that the main (may be the only) contribution into mutagenesis is made by monoadducts, whereas the lethal effect is conditioned by diadducts (cross-links). The comparison of the efficiency of mutagenic effects of 8-MOP+light with mutagenic effects of other kinds of irradiations was carried out. The possibility of repair of damaged 8-MOP+light phage sd DNA by transfection of Escherichia coli C (uvr+) and Cs (uvr-) lysozyme spheroplasts has been established. The repair mechanism of photodamage in intact phage sd induced with 8-MOP+light was also investigated using the method of two-step irradiation. It has been shown that 65% of photodamages are repaired in E. coli SK cells in the M9 medium, i. e. under cellular metabolism. The recovery of phage sd is completely inhibited in phosphate buffer. Unlike chloramphenicol (150 microgram/ml), 1% caffeine blocks the phage recovery only by 30%. The participation of phage sd determining enzymes in its intracellular recovery from 8-MOP+light damages is assumed.     

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.     

Excision repair does not protect Bacillus subtilis cells from inactivation by sunlight]

Exponentially growing Bacillus subtilis cells are highly sensitive to inactivating action of sunlight, the strain deficient in excision repair being every more sensitive than the uvr1 mutant. The inactivating effect is connected with the action of irradiation located in the left part of the spectrum (the whole UV region and some zones of the visible one). Increased sensitivity to sunlight disappeared when cells were exposed to sunlight in a liquid medium with casaminoacids (2 g/l). The inactivating effect was probably of photodynamic nature and was caused either by DNA lesions that were not removed by excision repair or by the damage which arose not in DNA.     

Effect of tsl (thermosensitive suppressor of lex) mutation on postreplication repair in Escherichia coli K-12.

Cells of Escherichia coli K-12 carrying lexA or recA mutations are more sensitive to UV radiation than corresponding wild-type cells and are defective in postreplication repair. Supressor mutations (tsl) have been described previously which increase the UV resistance of lexA uvr+, lexA uvrA, and recAI uvr+ strains, but not the resistance of recA1 uvrA strains. We have studied the effect of the tsl-1 mutation on postreplication repair and find that the enhanced survival conferred by this mutation is correlated with an increased capacity for postreplication repair.     

Postirradiation recovery dependent on the uvr-1 locus in Bacillus subtilis.

A mutant (uvr-1) of Bacillus subtilis that is deficient in excision of ultraviolet (UV)-induced pyrimidine dimers from deoxyribonucleic acid (DNA) shows a marked increase in ability to survive UV irradiation when plated on amino acid-supplemented agar medium compared with its survival ability when plated on nutrient plating medium, the effect is considered to be one of growth-dependent lethality. Irradiated stationary phase uvr-1 cells, incubated in liquid medium lacking amino acids required for growth, recover from this sensitivity to rich medium within 3 to 4 h after irradiation. Recovery is greatly reduced in the absence of glucose oiminated. Exponentially growing cells have a limited ability to recover from sensitivity to rich medium. Growth-dependent lethality can also occur in liquid medium. In nutrient broth the ability of irradiated stationary-phase uvr-1 cells to form colonies on defined agar medium decreases during postirradiation incubation, but treatmeth with chloramphenicol inhibits the loss of colony-forming ability. Recovery from sensitivity to rich media is inhibited by caffeine but not by 6-(p-hydroxyphenylazo)-uracil, and inhibitor of DNA replication. Alkaline sucrose gradient profiles show that conditions allowing recovery also favor maintaining intact DNA strands, whereas DNA strand breakage or degradation is associated with loss of viability. Recovery from sensitivity to rich medium has not been observed in the Ur+ parent or in strains carrying the mutations uvs-42 (another deficiency in dimer excision), recA1, or polA59. A uvr-1 recA1 mutants shows a higher level of recovery than does the recA1 single mutant, but a much lower level than the uvr-1 single mutant. Apparently, both the uvr-1 defect and Rec+ and PoII+ functions are essential for recovery from sensitivity to rich medium. For optimal recovery, growth immediately after irradiation must be delayed. The process requires energy, apparently involves recombination, and probably results in rejoining of DNA strands in which incision but not excision has occurred.     

Relative rates of repair of single-strand breaks and postirradiation DNA degradation in normal and induced cells of Escherichia coli.

Labeled DNA from irradiated Excherichia coli cells has been studied on an alkaline sucrose gradient without acid precipitation of the DNA. This enables the observation of both DNA repair and DNA degradation. The use of a predose of ultraviolet light (UV) causes induction of an inhibitor of postirradiation DNA degradation in lex+ strains. The effect of this induction on both the repair of single-strand breaks and DNA degradation has been followed in strains WU3610 (uvr+) and WU3610-89 (uvr-). The repair process is more rapid than the degradation, and when degradation is inhibited more repair is apparent. Cells that are lex- (Bs-1 and AB2474) cannot be induced for inhibition of degradation. Nevertheless, by observation at short times repair can be seen clearly. This repaired DNA is degraded, suggesting that the signal for DNA degradation is not a single-strand break.     

Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis

Repair of ultraviolet-irradiated transforming deoxyriboinucleic acid (DNA) in several strains of Bacillus subtilis was studied in order to determine the effects of excision repair and postreplication repair on transformation. Two mutations that cause a Uvr- and phenotype (uvr-1 and uvr-42) were shown to have strikingly different effects on repair of ultraviolet-irradiated transforming DNA. Genetic and kinetic evidence is presented to show that integrated DNA was apparently repaired by both excision and postreplication repair in wild-type and in uvr-1 recipients, although the latter excise pyrimidine dimers very slowly. In uvr-42 mutants, which are defective in incision at pyrimidine dimers, dimer-containing DNA was integrated. Postreplication repair apparently saved uvr-42 recipient cells from the lethal effects of integrated dimers, but the recombination events accompanying postreplication repair greatly reduced the linkage between closely linked genetic markers in the donor DNA. Repair of transforming DNA in a recG recipient, which does excision repair but not postreplication repair, was nearly as efficient as in wild-type cells. However, in this recipient linkage was altered only slightly, if at all, compared with wild-type cells. The apparent reduction in size of integrated regions of ultraviolet-irradiation transforming DNA probably results mainly from postreplication repair of larger integrated regions.     

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.     

Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells. Possible role of Dewar photoproducts in solar mutagenesis

In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0. 12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4)PP photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis.     

Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers

The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency. Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair. In E. coli the repair of UV-irradiated plasmid DNA is carried out only by the excision-repair system dependent on uvr genes. Treatment of UV-irradiated plasmid DNA with UV endonuclease from Micrococcus luteus greatly enhances the efficiency of transformation of E. coli uvr- mutants. Similar treatment with cell-free extracts of yeast rad1 mutant or wild-type strains as well as with nuclease BaL31, despite their ability for preferential cutting of UV damaged DNA, showed no influence on cell transformation.     

Protein oxidation,UVA and human DNA repair

Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency – with particular reference to NER and skin cancer risk.     

UV mutagenesis in E. coli with excision repair initiated by uvrABC or denV gene products

Mutation frequency responses produced by ultraviolet light are compared in 4 closely related strains of E. coli B/r having the same tyr(Oc) allele and different excision-repair capabilities: uvr+ (excision repair initiated by wild-type UvrABC activity), uvrA (excision repair defective), uvrA/pdenV-7 (excision repair initiated by endonuclease V of bacteriophage T4, DenV activity), and uvr+/pdenV-7 (excision repair initiated by UvrABC and DenV activities). The production of Tyr+ prototrophic mutants is classified into back-mutations and de novo or converted glutamine tRNA suppressor mutations to indicate different mutation events. Cells transformed with the plasmid pdenV-7 require larger exposures than the parent strains to produce comparable mutation frequency responses, indicating that DenV activity can repair mutagenic photoproducts. When damage reduction by UvrABC or DenV is compared for each of the specific categories of mutation, the results are consistent with the idea that pyrimidine dimers infrequently or never target back-mutations of this allele, frequently target the de novo suppressor mutations, and extensively or exclusively target the converted suppressor mutations. This analysis is based on the distinction that UvrABC-initiated excision repair recognizes dimer and non-dimer (pyrimidine (6-4) pyrimidone) photoproducts but that DenV-initiated repair recognizes only pyrimidine dimers.     

Gap-filling repair synthesis induced by ultraviolet light in a Bacillus subtilis Uvr- mutant.

Deoxyribonucleic acid repair synthesis was studied in one wild-type and two mutant strains of Bacillus subtilis that are defective in excision of pyrimidine dimers. The cells were irradiated with ultraviolet light, and 6-(p-hydroxyphenyl-azo)-uracil was used to block replicative synthesis, allowing only repair synthesis. One of the mutations (uvs-42) resulted in a severe inhibition of incision, dimer excision, and repair synthesis. In contrast, the other mutant (uvr-1) slowly incised and excised dimers and did repair synthesis in patches which appear to be several-fold longer than those in the wild-type strain, apparently because large gaps are produced at excision sites. The results indicate that the primary defect in uvs-42 cells is in initiation of dimer excision, whereas the uvr-1 mutation appears to be a defect in the exonuclease normally used to complete dimer excision.     

Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation

Exposure to solar UV radiation gives rise to mutations that may lead to skin cancer. UVA (320-340 nm) constitutes the large majority of solar UV radiation but is less effective than UVB (290-320 nm) at damaging DNA. Although UVA has been implicated in photocarcinogenesis, its contribution to sunlight mutagenesis has not been elucidated, and DNA damage produced by UVA remains poorly characterized. We employed HPLC-MS/MS and alkaline agarose gel electrophoresis in conjunction with the use of specific DNA repair proteins to determine the distribution of the various classes and types of DNA lesions, including bipyrimidine photoproducts, in Chinese hamster ovary cells exposed to pure UVA radiation, as well as UVB and simulated sunlight (lambda > 295 nm) for comparison. At UVA doses compatible with human exposure, oxidative DNA lesions are not the major type of damage induced by UVA. Indeed, single-strand breaks, oxidized pyrimidines, oxidized purines (essentially 8-oxo-7,8-dihydroguanine), and cyclobutane pyrimidine dimers (CPDs) are formed in a 1:1:3:10 ratio. In addition, we demonstrate that, in contrast to UVB and sunlight, UVA generates CPDs with a large predominance of TT CPDs, which strongly suggests that they are formed via a photosensitized triplet energy transfer. Moreover, UVA induces neither (6-4) photoproducts nor their Dewar isomers via direct absorption. We also show that UVA photons contained in sunlight, rather than UVB, are implicated in the photoisomerization of (6-4) photoproducts, a quickly repaired damage, into poorly repaired and highly mutagenic Dewar photoproducts. Altogether, our data shed new light on the deleterious effect of UVA.     

Heat and UV light resistance of vegetative cells and spores of Bacillus subtilis Rec-mutants.

The heat and UV light resistance of spores and vegetative cells of Bacillus subtilis BD170 (rec+) were greater than those of B. subtilis BD224 (recE4). Strain BD170 can repair DNA whereas BD224 is repair deficient due to the presence of the recE4 allele. Spores of a GSY Rec+ strain were more heat resistant than spores of GSY Rec- and Uvr- mutants. The overall level of heat and UV light resistance attained by spores may in part be determined by their ability to repair deoxyribonucleic acid after exposure to these two physical mutagens.     

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.     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light II. Effect of uvr Genes on Liquid Holding Recovery

The uvr mutations of Escherichia coli K-12 decrease the ability of cells to survive ultraviolet light (UV), to excise pyrimidine dimers from their deoxyribonucleic acid and to reactivate bacteriophage exposed to UV. The rec mutations decrease the ability of the cells to survive UV and to undergo genetic recombination. Certain rec mutations, including recA1, rec-12, recA13, and rec-56, are necessary for the expression of liquid-holding recovery (LHR), observed as an increase in colony-forming ability when irradiated cells are held in buffer in the dark. These rec mutations appear to act indirectly to permit the detection of LHR rather than to affect its occurrence directly. We have tested the effect of uvr markers on LHR in cells containing one of these rec mutations. Recombinants containing rec-56 together with a uvr marker were constructed and tested for LHR. None of the 39 recombinants examined, carrying uvrA6, uvrB5, or uvrC34, showed LHR. Three rec(-)uvr(-) strains were also tested for photoreactivation. In all three, photoreactivation was observed, indicating that they contained detectable amounts of pyrimidine dimers. Our results are consistent with the idea that uvr mutations inactivate LHR, and suggest that LHR reflects excision-dependent repair of pyrimidine dimers.     

Mutations induced by ultraviolet light

The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms involving oxidized DNA bases. We also discuss the role of DNA damage-tolerant DNA polymerases in UV lesion bypass and mutagenesis.