Excision Repair Characteristics of recB−res− and uvrC− Strains of Escherichia coli

An Escherichia coli strain carrying the recB21 and res-1 mutations showed an abnormally low level of colony-forming ability although it grew essentially normally in liquid medium. The recB21 res-1 strain showed little, if any, of the ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown characteristic of the res-1 mutant. Nevertheless, the double mutant was far more sensitive to UV than either the res-1 or the recB21 strain. When compared with a wild-type strain, the rate of release of dimers from UV-irradiated DNA was very slow in the recB21 res-1, but normal in the res-1 recB(+) or recB21 res(+) mutants. However, the ratio of dimer-to-thymine released into the acid-soluble fraction was three times higher than the wild type in recB21 res(+) and recB21 res-1 and only one-tenth as high as the wild type in res-1 rec(+). Alkaline sucrose gradient centrifugation revealed occurrence of single-strand incision of UV-irradiated DNA and the restitution of nicked DNA at a similar rate in the recB21 res-1 and recB21 res(+) strains. Mutants uvrC(-) showed increased amounts of nicks in their DNA with increasing incubation time after UV irradiation, although no detectable amounts of dimers were excised from UV-irradiated DNA. From these results, it is concluded that the increased sensitivity of the res-1 strain to UV light is due to a reduced ability to excise dimers from UV-irradiated DNA and that the high rate of UV-induced breakdown of DNA is not the primary cause. A possible role of uvrC gene in the excision repair is discussed.     

Fate of thymine-containing dimers in the deoxyribonucleic acid of ultraviolet-irradiated mutator T1 Escherichia coli transfuctants

In order to determine whether a relationship generally exists between the mutator property (mutT1) and repair of ultraviolet (UV) irradiation damaged DNA, we performed spontaneous mutation rate and UV-survival determinations without and with acriflavin (4 μg/ml) in P1 phage mediated mut T1 Escherichia coli transductants. The strains constructed were assumed to be cosigenic except for the mutator factor. The mutT1 uvrA, uvrB or exrA transdunctants had mutation rates similar to the donor strain. Double mutants containing mutT1 and uvrB or exrA had the same level of UV survival as the parent with the same mutator phenotype. Mutator strains were normal for host-cell reactivation of UV-irradiated phage T1, and phage lambda was UV-inducible. The fate of UV-induced thymine-containing dimers in the deoxyribonucleic acid (DNA) of mutT1 transductants was investigated. Dark repair of pyrimidine dimers is equally sensitive in the nonmutator and mutator Hcr⁺. During incubation in the dark, dimers were excised to the same extent from the DNA of the Hcr⁺ mutator and nonmutator transductants but remained in the DNA of the Hcr^? mutant.     

Excision Repair of Uv Radiation-Induced DNA Damage in Caenorhabditis Elegans

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts [cyclobutane dimers and (6-4) photoproducts]. Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.     

Role of pyrimidine dimer excision in loss of potential streptomycin resistance mutations of ultraviolet-irradiated Escherichia coli on phosphate-buffered agar.

The frequency of ultraviolet (UV)-induced mutations to streptomycin resistance dropped rapidly when starved Escherichia coli strains WP-2 B/r and B/r T- were incubated on phosphate-buffered agar (PBA), but was reduced only slightly in a WP-2 hcr- mutant. During postirradiation, incubation viability remained approximately constant. Cells given an optimal recovery treatment with photo-reactivating light showed no further recovery if subsequently incubated on PBA. At least 70% of the mutations induced to streptomycin resistance by UV could be repaired. The loss of potential streptomycin-resistant mutants was markedly reduced in strain B/r T- when 5 mug of acriflavin or 700 mug of caffeine per ml was added to PBA. The excision of UV-induced thymine-containing dimers from E. coli tb/r T- was investigated. Dimer excision progressed more slowly when the cells were incubated on PBA containing acriflavin or caffeine. There was no congruity between the kinetics of dimer excision and the kinetics of mutant loss. Our results indicate that removal of potential streptomycin-resistant mutants is considerably faster than the excision of pyrimidine dimers.     

Excision repair of ultraviolet damage in mammalian cells. Evidence for two steps in the excision of pyrimidine dimers

The incidence of pyrimidine dimer formation and the kinetics of DNA repair in African green monkey kidney CV-1 cells after ultraviolet (UV) irradiation were studied by measuring survival, T4 endonuclease V-sensitive sites, the fraction of pyrimidine dimers in acid-insoluble DNA as determined by thin layer chromatography (TLC), and repair replication. CV-1 cells exhibit a survival curve with extrapolation number n = 7.8 and Do = 2.5 J/m2. Pyrimidine dimers were lost from acid-insoluble DNA more slowly than endonuclease-sensitive sites were lost from or new bases were incorporated into high molecular weight DNA during the course of repair. Growth of CV-1 cultures in [3H]thymidine or X-irradiation (2 or 10 krads) 24 h before UV irradiation had no effect on repair replication induced by 25 J/m2 of UV. These results suggest that pyrimidine dimer excision measurements by TLC are probably unaffected by radiation from high levels of incorporated radionuclides. The endonuclease-sensitive site and TLC measurements can be reconciled by the assumption that pyrimidine dimers are excised from high molecular weight DNA in acid-insoluble oligonucleotides that are slowly degraded to acid-soluble fragments.     

Repair of Single-Strand Deoxyribonucleic Acid Breaks in Ultraviolet Light-Irradiated Haemophilus influenzae

The wild-type strain and mutants of Haemophilus influenzae, sensitive or resistant to ultraviolet light (UV) as defined by colony-forming ability, were examined for their ability to perform the incision and rejoining steps of the deoxyribonucleic acid (DNA) dark repair process. Although UV-induced pyrimidine dimers are excised by the wild-type Rd and a resistant mutant BC200, the expected single-strand DNA breaks could not be detected on alkaline sucrose gradients. Repair of the gap resulting from excision must be rapid when experimental conditions described by us are employed. Single-strand DNA breaks were not detected in a UV-irradiated sensitive mutant (BC100) incapable of excising pyrimidine dimers, indicating that this mutant may be defective in a dimer-recognizing endonuclease. No single-strand DNA breaks were detected in a lysogen BC100(HP1c1) irradiated with a UV dose large enough to induce phage development in 80% of the cells.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Role of DNA damage in the inactivation of Saccharomyces cerevisiae yeasts by 313-nm ultraviolet light

The relative contribution of respiration photoinhibition and DNA damage in the lethal effect induced by 313 nm ultraviolet light (UV) has been investigated in some strains of the yeast Saccharomyces cerevisiae. It has been shown that cells inactivation is essentially due to photo-induced damage to DNA. By photoreactivation experiments it has been found that dimers of the pyrimidine bases are the main lethal photoproducts induced in the DNA by 313 nm ultraviolet light.     

Action of T4 endonuclease V on DNA containing various photoproducts

The action of T4 endonuclease V on DNA containing various photoproducts was investigated. (1) The enzyme introduced strand breaks in DNA from ultraviolet-irradiated vegetative cells of Bacillus subtilis but not in DNA from irradiated spores of the same organism. DNA irradiated with long wavelength (360 nm peak) ultraviolet light in the presence of 4,5',8-trimethylpsoralen was not attacked by the enzyme. These results indicate that 5-thyminyl 5,6-dihydrothymine (spore photoproduct) and psoralen mediated cross-links in DNA are not recognized by T4 endonuclease V. (2) DNA of phage PBS1, containing uracil in place of thymine, and DNA of phage SPO1, containing hydroxymethyluracil in place of thymine, were fragmented by the enzyme when the DNA's had been irradiated with ultraviolet light. T4 endonuclease V seems to act on DNA with pyrimidine dimers whether the dimers contain thymine residues or not.     

Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts

To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of hypochlorite on spores of Bacillus subtilis lacking small acid-soluble proteins

M.Z.H. SABLI, P. SETLOW AND W.M. WAITES. 1996. α/β-Type small acid-soluble proteins (SASP) bind to spore DNA and protect it against ultraviolet light, heat, hydrogen peroxide and freeze drying, making the spores much more resistant than vegetative cells to these agents. Spores of a mutant of Bacillus subtilis lacking the two major α/β-type SASP were almost 30 000-fold less resistant to hypochlorite than were wild-type spores. After treatment with hypochlorite, surviving spores of the mutant, but not those of the wild type, showed higher levels of mutation, suggesting that SASP contribute to hypochlorite resistance by protecting spore DNA.     

Integration and Repair of Ultraviolet-Irradiated Transforming Deoxyribonucleic Acid in Haemophilus influenzae

The extent of association between donor transforming deoxyribonucleic acid (DNA) and recipient DNA in Haemophilus influenzae as a function of ultraviolet (UV) dose to the transforming DNA has been measured by isopycnic analysis of lysates of (3)H-labeled recipient cells exposed to DNA labeled with (32)P and heavy isotopes. Except for doses above 15,000 ergs/mm(2), the results of these measurements are in good agreement with previous estimates made by another technique. Experiments with a mutant temperature sensitive for DNA synthesis and another mutant defective in excision of pyrimidine dimers suggest that the discrepancy between the methods of high doses results from DNA synthesis, in which portions of the associated donor DNA containing pyrimidine dimers are excised and broken down, and the components are reutilized for synthesis.Repair of UV-irradiated, transforming DNA during incubation of recipient cells is observed as an increase in transforming ability when fractions from CsCl gradients of cell lysates are assayed on excision-deficient cells. When transforming DNA containing markers of different UV sensitivities is used, repair of the UV-resistant nov marker by excision proficient cells takes place exclusively in the donor DNA that is associated with recipient DNA, and this repair is observed even in the absence of DNA synthesis. However, no repair is observed in the case of the more UV-sensitive str marker, possibly because excision events may remove a large fraction of the integrated str markers in addition to repairing a small fraction of the integrated DNA containing this marker.     

Deoxyribonucleic acid excision repair in chromatin after ultraviolet irradiation of human fibroblasts in culture.

We have exposed confluent normal human fibroblasts to ultraviolet (UV) fluences of 5, 14, or 40 J/m2 and monitored the specific activity of post-UV repair synthesis in chromatin with [3H]thymidine pulses. We have shown that under conditions where no semiconservative deoxyribonucleic acid (DNA) synthesis is detectable, the specific activity of repair label in micrococcal nuclease resistant (core particle) DNA is about one-fifth that in bulk DNA at all three UV fluences. On the other hand, the distribution of thymine-containing pyrimidine dimers in bulk and nuclease-resistant regions measured either immediately after irradiation or at later times showed no significant differences; preferential labeling of linker (nuclease-sensitive) DNA during repair synthesis is thus apparently not due to a predominance of UV-induced photoproducts in linker relative to core particle DNA in the nucleosome. Pulse and pulse--chase experiments at 14 or 40 J/m2 with normal human or repair-deficient xeroderma pigmentosum (XP) cells showed that at most 30% of repair label in all these cells shifts from nuclease-sensitive (linker) DNA to nuclease-resistant (core particle) DNA.     

Effect of oxygen on liquid holding recovery of Bacteroides fragilis.

Liquid holding recovery (LHR) in ultraviolet-irradiated Bacteroides fragilis cells occurred under aerobic conditions but was inhibited by anaerobic conditions. The increase in survival after aerobic LHR resulted in an increase in the shoulder regions of the ultraviolet survival curves. Maximum LHR was obtained after holding the cells for 2 to 3 h. LHR was temperature dependent, and in stationary-phase cells LHR was independent of nutrients. Higher levels of LHR occurred in exponential-phase cells than in stationary-phase cells, and LHR was affected by nutrients in exponential-phase cells. Sublethal concentrations of caffeine and acriflavine inhibited LHR. In addition to LHR, minimal medium recovery also occurred in the concentration of [3H]thymine-containing dimers in the acid-insoluble fraction of the cells. A corresponding increase in [3H]thymine-containing dimers was observed in the acid-soluble fraction after LHR. Although a small proportion of irradiated cells produced filaments, this phenomenon was not directly related to LHR in B. fragilis.     

Repair of deoxyribonucleic acid in ultraviolet light-sensitive and -resistant Dictyostelium discoideum strains.

Some responses of the cellular slime mold Dictyostelium discoideum to ultraviolet light (UV) irradiation were investigated by analyzing two aspects of deoxyribonucleic acid (DNA) excision repair in the vegetative cells: (i) the fate of thymine-containing dimers and (ii) the production and rejoining of single-strand breaks. Experiments were done with the parental, radiation-resistant NC-4 strain and with the radiation-sensitive gammas-13 strain. The majority (greater than 85%) of the thymine-containing dimers produced in both strains by an energy fluence of 100/Jm2 were removed from the acid-insoluble DNA fraction within the first 3 to 4 h of reincubation in the dark. Moreover, as measured by alkaline sucrose gradients, single-strand breaks appeared in the DNA of both NC-4 and gammas-13 irradiated cells very rapidly and at low temperatures. This was presumed to be a result of the incision (nicking) step of excision repair as performed by UV-specific endonuclease(s). In NC-4 the time required for dimer excision correlated with the sealing of breaks as well as with the UV-induced division delays. In gammas-13 the single-strand breaks were closed at a slower rate than in NC-4. However, this was not accompanied by more extensive division delays.     

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.     

Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts

We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone. The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV. Furthermore, the treatment of UV-irradiated DNA with photolyase from E. coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA. A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts. These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA. Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells. Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.     

Nonrandom induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin

Radioimmunoassays that detect pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane dimers were used to determine the relative induction of these photoproducts in nucleosomal (core) and internucleosomal (linker) DNA in human cell chromatin irradiated with UV light. Cyclobutane dimers were formed in equal amounts/nucleotide in core and linker DNA, whereas (6-4) photoproducts occurred with 6-fold greater frequency/nucleotide in linker DNA.     

The fate of thymine-containing dimers in ultraviolet-irradiated Halobacterium cutirubrum

Extremely halophilic bacteria do not recover in the dark from the effects of ultraviolet radiation, although they can be completely photoreactivated, thus proving that pyrimidine dimers are the cytotoxic photoproducts. We have now shown, by studying the fate of thymine-containing dimers during dark liquid-holding of Halobacterium cutirubrum, that the lack of dark repair in these bacteria is due to their inability to excise such lesions, in contrast to Escherichia coli B. Nevertheless, extensive nonspecific degradation of DNA occurs in H. cutirubrum following ultraviolet irradiation, so the lack of dimer excision is not due to a generalised inability to degrade DNA after such treatment. These findings raise interesting questions concerning the basis of the resistance of halophiles to ultraviolet radiation.     

Photosensitization of DNA by dipicolinic acid, a major component of spores of Bacillus species.

The DNA in spores of Bacillus species exhibits a relatively novel photochemistry, as 5-thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) is by far the major UV photoproduct whereas cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the major photoproducts in growing cells. Dehydration and more importantly complexation of DNA by alpha/beta-type small, acid-soluble spore proteins (SASP) have been shown to partly explain the photochemistry of spore DNA. The large amount ( approximately 10% of dry weight) of the spore's dipicolinic acid (DPA) also has been shown to play a role in spore DNA photochemistry. In the present work we showed by exposing spores of various strains of B. subtilis to UVC radiation that DPA photosensitizes spore DNA to damage and favors the formation of SP. The same result was obtained in either the presence or absence of the alpha/beta-type SASP that saturate the spore chromosome. Addition of DPA to dry films of isolated DNA or to frozen solutions of thymidine also led to a higher yield of SP and increased ratio of CPDs to 6-4PPs; DPA also significantly increased the yield of CPDs in thymidine exposed to UVC in liquid solution. These observations strongly support a triplet energy transfer between excited DPA and thymine residues. We further conclude that the combined effects of alpha/beta-type SASP and DPA explain the novel photochemistry of DNA in spores of Bacillus species.