A STUDY OF THE RELATIONSHIP BETWEEN SURVIVAL AND REPAIR SYNTHESIS'' OF DNA AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. and the postirradiation repair synthesis of E. coli 15 T^- 555-7 thy meth arg trp and E. coli B/r (HCR⁺) was followed. Prestarvation increased the number of survivors about 30–100 fold in both strains at doses 600-1200ergs/mm². In contrast to survival no increase in repair synthesis was observed. Thus, the increase in survival has to be brought about by a mechanism which seems to be independent of additional repair synthesis.     

PATTERNS OF NUCLEIC ACID AND PROTEIN SYNTHESIS DURING MODIFICATION OF LETHAL ULTRAVIOLET LESION

Abstract— We have shown previously that the viability of E. coli B.U.^- and E. coli B. try^-could be restored after u.v.-irradiation if post-irradiation incubation took place in the presence of chloramphenicol or in the absence of an essential metabolite. By way of contrast, the viability of the u.v.-irradiated E. coli K.₁₂ meth^- decreased rapidly during post-irradiation incubation in the presence of chloramphenicol or in the absence of methionine. During the study of the biosynthesis of nucleic acids and protein in the above strains we made the following observations.
Protein synthesis was permanently inhibited in both amino acid requiring strains during post-irradiation incubation in the absence of the respective essential metabolite. In E. coli B.U.^- a slow synthesis of protein could be observed even if post-irradiation incubation took place in the absence of uracil.
DNA synthesis was completely and permanently inhibited in both E. coli B. try^- and E. coli K.₁₂ meth^- during post-irradiation incubation in the absence of the respective essential amino acid. In E. coli B.U.^-, however, DNA synthesis resumed after a prolonged lag even if the bacteria were incubated after u.v.-irradiation in the absence of uracil.
RNA synthesis was completely and permanently inhibited in both E. coli B. mutants, the uracil requiring and the tryptophan requiring, if they were incubated after irradiation in the absence of uracil and tryptophan respectively. In E. coli K.₁₂ meth^- a small amount of RNA is synthesized during post-irradiation incubation in the absence of the essential amino acid.     

THE U.V. SENSITIVITY OF BACTERIA: ITS RELATION TO THE DNA REPLICATION CYCLE

Abstract— A striking increase in the shoulder of the u.v. survival curve but no change in the limiting slope is obtained when cultures of Escherichia coli strain TAU complete the DNA replication cycle in the absence of concommitant protein synthesis prior to irradiation. The u.v. sensitivity of protein synthesis or RNA synthesis is not altered significantly by this treatment.
In contrast to the result for strain TAU, there is no significant change in the u.v. survival curve for the u.v. sensitive E. coli B_s-1 when its DNA replication cycle is completed under similar conditions.
Following a period of inhibited protein synthesis there is a delay in the reinitiation of the normal DNA replication cycle when protein synthesis resumes. This delay would allow time for an intracellular repair system to operate before the attempted resumption of normal replication. Strain B_s-1, which is deficient in this repair system, would not be expected to benefit from such a delay, as consistent with the observed results. A model is presented to account for lethality due to attempted DNA replication during a period of repair synthesis. The maximum survival for a given u.v. dose would be predicted for a culture which has completed the normal DNA replication cycle prior to irradiation and which is not permitted to reinitiate the cycle until all possible repair synthesis is completed.     

THE ROLE OF HOST-CELL REPAIR IN LIQUID-HOLDING RECOVERY OF U.V.-IRRADIATED ESCHERICHIA COLI

Abstract— The experiments reported give evidence that liquid-holding recovery (LHR) of u.v. irradiated E. coli cells involves basically the same type of dark repair which causes reactivation of phage and which results in much increased survival of the cells themselves [host-cell reactivation (HCR)]. LHR is very small in the two HCR(-) strains B syn^- and B_s-1, but occurs to larger but different extents in the three HCR(+) strains B, B/r, and B/r (Λ). LHR is inhibited if the liquid contains caffeine or acriflavine, both of which are known to inhibit HCR. The results indicate that most of the LHR effect, if not all, occurs during the liquid holding, rather than under growth conditions after liquid holding. It is assumed that the holding itself allows a prolonged time for, and therefore an enhancement of, HCR. It is thus implicit that LHR can be observed only where otherwise HCR of repairable u.v. damage would be incomplete, and that different extents of LHR, as observed in the three HCR(+) strains, reflect different extents of incompleteness of HCR. It is concluded that the repairable u.v. hits which are not fully repaired by HCR are predominantly those concerned with the extra u.v. sensitivity of the strains B and B/r (Λ), relative to B/r.     

REPAIR OF UV DAMAGE IN ESCHERICHIA COLI UNDER NON-GROWTH CONDITIONS

Abstract. –A large difference in survival occurs between buffered suspensions of E. coli irradiated with UV radiation at a low fluence rate and those irradiated at a high fluence rate. For sufficiently large fluences, the extent of this fluence rate dependent recovery (FRR) is about two orders of magnitude greater than that which can be brought about by liquid holding recovery (LHR) following high fluence rate irradiation in most of the E. coli strains studied. LHR and FRR occur in excision resynthesis repair proficient (ERR⁺) but not ERR^- strains of E. coli , although its observation can be masked in strains with complete repair potential upon subsequent growth on nutrient plates. Accumulation of DNA strand interruptions and excision of cyclobutyl dipyrimidine occur during LHR and FRR but are more extensive for the latter. Our data suggest mat events beyond incision and excision occur during LHR and FRR, but differences in the extent of ERR during LHR and FRR cannot account for the difference in cell survival between these two phenomena.     

RELATIONSHIP BETWEEN SURVIVAL AND THYMINE-DIMER EXCISION IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. light and excision of thymine dimers of bacterial strains E. coli B/r hcr ⁺ thy- trp ^-, E. coli B/r hcr ^-thy^- trp ^-, and E, coli 15 T^- 555–7 thy ^- meth ^- trp ^- arg ^- has been studied.
The prestarvation increased the resistance of all the strains but reasonably inhibited excision of thymine dimers. Thus the enhancement of u.v. resistance after amino acid prestarvation was not due to more complete excision of thymine dimers.     

REC A +-DEPENDENT SYNERGISM BETWEEN 365 NM AND IONIZING RADIATION IN LOG-PHASE ESCHERICHIA COLI: A MODEL FOR OXYGEN-DEPENDENT NEAR-UV INACTIVATION BY DISRUPTION OF DNA REPAIR

Abstract —The oxygen dependence of 365 nm inactivation of colony-forming ability of Escherichia coli has been investigated in two series of DNA repair-deficient K12 mutants grown to mid-exponential phase. All strains except a uvr A rec A double mutant are more sensitive to inactivation under O₂ and show a lower threshold dose. The inactivation of photoreactivating enzyme in a crude cell extract and DNA repair disruption are both reduced when irradiation is carried out under nitrogen. The rec A gene-dependent synergism between 365 nm and ionising radiation is reversible if cells are incubated in full growth medium before ionising radiation treatment. In a wildtype strain, incubation for 2.5 h in full growth medium after 10⁶ J m^-2 365 nm radiation changes a sensitised response to a protection from ionising radiation. Protection is not seen at 1.5 times 10⁶ J m^-2. A tentative model for near UV lethality in logarithmic phase cells is suggested which proposes two classes of lesions. One requires oxygen for it's induction, is rapidly fixed as a lethal event as a result of repair disruption, and is primarily responsible for cell death after aerobic 365 nm irradiation. The other lesion, possibly pyrimidine dimers, may lead to cell death under anaerobic conditions.     

EFFECT OF PHOTOREACTIVATING LIGHT ON LETHAL AND PRE-MUTATIONAL UV LESIONS IN ESCHERICHIA COLI WP2s CARRYING THE R46 MUTATOR PLASMID

Abstract—An excision-deficient E. coli strain carrying the R46 mutator plasmid showed a different response towards photo-reactivation after UV irradiation than the same strain without plasmid. While the photoreactivation of lethal lesions was comparable in both strains, the number of UV-induced mutants per 10⁶ survivors was slightly reduced for the plasmid bearing strain by photoreactivating light at UV fluences below 60 mJ/m² but increased at higher fluences. To explain this it is proposed that some UV photoproduct(s) of DNA other than cyclobutane dipyrimidine dimers are pre-mutational lesions for error-prone DNA repair by the plasmid, P-repair, but not for SOS-repair.     

LIQUID HOLDING RECOVERY OF PHOTODYNAMIC DAMAGE IN E. COLI

Escherichia coli cells lose viability when irradiated with visible light in the presence of acridine dyes. There has been some controversy about whether such photodynamic damage can be repaired. Several investigations on different E. coli strains failed to reveal any significant difference in the sensitivity to photodynamic damage between radiation-resistant E. coli B/r and radiation-sensitive E. coli B_s-1 strains (Uretz, 1964; Janovska et al. , 1970) suggesting the absence of repair. On the contrary, other investigators (Rupp, 1966; Harm, 1968) strongly predicted the existence of a dark-repair mechanism for such damage. No systematic study of liquid-holding recovery of photodynamic damage has yet been reported and knowledge of repair capacity for photodynamic damage is still incomplete. In the present communication, liquid-holding-repair capability of E. coli B/r and B_s-1 for photodynamic inactivation in the presence of acridine orange or acriflavine has been investigated. These studies show that B/r can repair photodynamic damage while B_s-1 is deficient in this ability.     

DECREASED SURVIVAL RESULTING FROM LIQUID-HOLDING OF U.V.-IRRADIATED ESCHERICHIA COLI C AND SCHZZOSACCHAROMYCES POMBE

Abstract— Ultraviolet-irradiated cells of E. coli C and of haploid wild type yeast Schizosac-charomyces pombe , held in buffer at 22°-25°C for various periods of time prior to plating, show a lower survival than those plated immediately after irradiation. This 'negative liquid-holding effect' (NLHE) contrasts 'liquid-holding recovery' (LHR), found in a number of other E. coli strains and in Saccharomyces cerevisiae . NLHE was observed at all u.v. doses tested. The effect is maximal at holding temperatures in the range 25–30°C, it is very small at 5°C and (in E. coli C) at 44°C. NLHE and LHR resemble each other in several respects. In E. coli both effects are inhibitable by the dark repair inhibitors acriflavine, caffeine and potassium cyanide. They do not occur in nutrient broth, and they are much reduced if the irradiated cells were illuminated with photoreactivating light before holding. NLHE in S. pombe shows characteristics similar to those observed in E. coli C . Mutations leading to increased u.v. sensitivity in E. coli C and S. pombe can alter the liquid-holding response so that LHR is observed. Tetrad analysis of crosses between u.v.-sensitive and u.v.-resistant S. pombe strains indicates that a single chromosome region can control both u.v. sensitivity and liquid-holding response. Several possibilities explaining NLHE are discussed. From current knowledge about dark repair processes and from the similarities between NLHE and LHR in E. coli it seems likely that the two effects reflect slight changes in the efficiency of dark repair.     

THYMINE DIMERIZATION IN L-STRAIN MAMMALIAN CELLS AFTER IRRADIATION WITH ULTRAVIOLET LIGHT AND THE SEARCH FOR REPAIR MECHANISMS

Abstract— The formation of thymine dimers in the DNA of L -strain mammalian cells after irradiation with ultraviolet light has been demonstrated. The amount of dimer formed rises with the dose of u.v. light.
In the course of post-irradiation incubation the thymine dimers remain in the TCA insoluble fraction and diminish as did the other thymidine-H³ derivatives with increasing incubation time. The dimer is not found in the soluble fraction. Thus, dimer excision (i.e. its liberation into the soluble fraction) as an expression of repair of radiation damage analogous to dark repair in E. coli was not found in these experiments.     

DNA SYNTHESIS-KINETICS, CELL DIVISION DELAY, AND POST-REPLICATION REPAIR AFTER UV IRRADIATION OF FROZEN CELLS OF E. COLI B/r

Abstract— Previous studies have shown that the relative yields of photoproducts produced in the DNA of Escherichia coli cells UV irradiated at -79°C differ from those produced at +21°C; the yield of DNA-protein cross-links was markedly enhanced at -79°C while the yield of thymine dimers was reduced. In the present studies, cells of E. coli B/r thy were frozen at -79°C, and then UV irradiated (254nm) while frozen(4.7 J m^-2), or after thawing (22 Jm^-2). Essentially the same survival, cell division delay, and DNA synthesis kinetics were observed for these two samples after irradiation, even though the UV fluence differed by a factor of ˜5. This supports previous observations that a correlation exists between the magnitude of the effects of UV radiation upon DNA synthesis kinetics and on cell survival. The weight average molecular weight of the pulse labeled DNA in the sample irradiated at +21°C was one-half that of the sample irradiated at -79°C, and complete repair of daughter-strand gaps was observed in both cases. Thus, UV-induced lesions produced in cells at -79°C (i.e. DNA-protein cross-links) appear to be amenable to post-replicational repair. While the overall DNA synthesis kinetics were the same for the two irradiation procedures, the apparent number of lesions produced per unit length of DNA was not. This suggests that each of the lesions produced in frozen cells, although apparently fewer in number, must cause a longer local delay in DNA synthesis than those lesions produced at +21°C.     

QUANTITATIVE EVIDENCE FOR ENZYMATICALLY-INDUCED DNA DOUBLE-STRAND BREAKS AS LETHAL LESIONS IN UV IRRADIATED pol+ AND polAl STRAINS OF E. COLI K-12

Abstract— We have recently reported that DNA double-strand breaks arise enzymatically during the course of excision repair in uvr ⁺ strains of Escherichia coli K-12. Survival curves for ultraviolet (UV) irradiated E. coli K-12 pol⁺ (JG139) and polA1 (JG138) strains have a pronounced shoulder region. The regions of the survival curves at which killing approaches exponential correspond to the fiuences at which DNA double-strand breaks (assumed to be lethal events) accumulate linearly. Reducing the number of UV photoproducts either by photoreactivation or fluence fractionation results in an increase in survival and a decrease in the yield of DNA double-strand breaks in both strains. These data support the hypothesis that enzymatically-induced DNA double-strand breaks may be the lesion ultimately responsible for UV-induced cell killing in the pol⁺ strain of E. coli K-12. and perhaps also in the polA1 strain.     

PROTECTION OF ESCHERICHIA COLI CELLS AGAINST THE LETHAL EFFECTS OF ULTRAVIOLET AND X IRRADIATION BY PRIOR X IRRADIATION: A GENETIC AND PHYSIOLOGICAL STUDY

Abstract— When log phase cells of wild-type E. coli K-12 were maintained in growth medium after X irradiation, they became progressively more resistant to a subsequent exposure to UV or X radiation. The time to achieve maximum resistance was about 60 min. The uvrB, uvrD, polA and certain exrA strains (W3110 background) also demonstrated this X ray-induced resistance to subsequent UV or X irradiation but recA, recB, lex (AB1157 or W3110 backgrounds) and other exrA strains (AB1157 background) did not. The resistance induced in wild-type, uvrB and uvrD cells was characterized by the production or enhancement of a shoulder on the survival curves obtained for the second irradiation, while the resistance induced in the W3110 exrA strains was expressed only as a change in slope. The induction of resistance in the W3110 exrA strain was not inhibited by the presence of chloramphenicol, but that in the wild-type cells appeared to be. The production or enhancement of a shoulder on the survival curves of the rec ⁺ lex ⁺ exr ⁺ cells is consistent with the concept of the radiation induction of repair enzymes. Alternative explanations, however, are discussed.     

DEGRADATION OF THE DNA OF ESCHERICHIA COLI K12 REC- (JC1569b) AFTER IRRADIATION WITH ULTRAVIOLET LIGHT*

Abstract— Degradation of the DNA of a rec^- mutant of Escherichia coli K12 (JC1569 b) induced by u.v. light was investigated. The rate of degradation was much larger by growing bacteria than by stationary cells. When growing bacteria were starved for amino acids, their DNA became resistant to irradiation. The mode of u.v.-induced degradation was investigated by comparing the time course of release from the acid-insoluble fraction of the label for two growing cultures; the one was pulse-labeled with ³H-thymidine and the other was pulse-labeled and chased thereafter for 12 min. It was found that the label incorporated into the former culture begins to be lost from the acid-insoluble fraction prior to the loss of the label incorporated into the latter culture. It was concluded that breakdown of the replicating point precedes degradation of the bulk of the DNA. This result suggested that the replicating point is a sensitive site to irradiation and the u.v.-induced degradation of DNA seemed to be influenced by the state of chromosome at the time of irradiation. Experiments of centrifugation of lysed spheroplasts of bacteria uniformly labeled with ³H-thymidine in alkaline sucrose demonstrated that DNA of low molecular weight appeared after irradiation with only 5 ergs/ mm², and that the molecular weight could not be restored by post-irradiation incubation. Considering these results, an hypothesis is proposed concerning the initiation of induced degradation of the DNA of the rec^- mutant.     

ULTRAVIOLET RADIATION INHIBITS BOTH ACCESSORY CELL AND RESPONDER T CELL FUNCTION IN THE PROLIFERATIVE RESPONSE TO HAPTENATED-SELF

Abstract— Photoprotection i. e. the increased resistance of the cells preilluminated with near ultraviolet light (300–380 nm) to the lethal action of 254 nm radiations is observed in wild-type Escherichia coli B cells (which exhibits the Fil phenotype) but requires either an integrated prophage or a rec A mutation to be detected in E. coli K12 strains. Here we have demonstrated that significant photoprotection occurs in an E. coli K12 rec A⁺ cell containing the Ion allele which is responsible for filamentous growth (Fil phenotype) after 254 nm irradiation. The Fil phenotype can be suppressed by the sfi A of sfi B suppressor genes. Since the E. coli K12 rec A⁺ Ion sfi B strain exhibits no more photoprotection, these data support the conclusion that in Ion strains photoprotection is due to the abolition of the 254 nm induced filamentation by the near ultraviolet treatment. In addition, we show here that near ultraviolet illumination of the cells leads to a severe restriction of the bulk protein synthesis, as well as of the inducibility of β-galactosidase and tryptophanase. These effects are observed only in nuv ⁺ cells that contains 4-thiouridine the chromophore responsible for photoprotection. We propose that in Ion (lysogenic) strains, photoprotection is due to prevention of the SOS response. During the growth lag, the low residual level of protein synthesis does not allow the induction of the SOS response and accordingly prevents filamentation (the lytic cycle). Concomitantly the SOS triggering signals are eliminated via DNA repair.     

LETHAL INTERACTION BETWEEN ULTRAVIOLET-VIOLET RADIATIONS AND METHYL METHANE SULPHONATE IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI

Abstract— The lethal interaction between monochromatic radiation at various wavelengths and methyl methane sulphonate was tested in strains of Escherichia coli proficient and deficient in DNA repair. In the repair proficient wild-type strain K12 AB1157, the efficiency of sensitization to MMS as a function of dose (at 334 nm, 365 nm and 405 nm) was found to be directly correlated with the dose necessary to remove the shoulder from the survival curve at the wavelength employed. The 365 nm: MMS interaction was also observed in other repair proficient E. coli strains (W3110 and B/r) but was absent in a recA and a polA strain. Pre-treatment of AB1157 with MMS leads to a much larger interaction than pre-irradiation with 365 nm. It is concluded that dose-dependent damage to DNA repair by the near-UV radiation is involved in the interaction and possibly that MMS causes irreversible damage 10 repair enzymes.     

SOME OBSERVATIONS ON THE LETHAL EFFECTS OF NEAR-ULTRAVIOLET LIGHT ON ESCHERICHIA COLI, COMPARED WITH THE LETHAL EFFECTS OF FAR-ULTRAVIOLET LIGHT

Abstract— Near-ultraviolet light (365.5 nm) reduces the ability of Escherichia coli B/r and B_8-1, to form colonies on nutrient agar after irradiation. This lethal effect is distinct from that obtained after far-u.v. irradiation (253.7 nm) because the far-u.v. sensitive and resistant strains are equally susceptible to near-u.v. Variation in susceptibility to ultraviolet light during growth is more marked for near-u.v. than for far-u.v. The number of survivors after near-u.v. irradiation of log phase cells is affected by several post-irradiation treatments; more cells survive if growth immediately after irradiation occurs at higher temperatures (unlike far-u.v.). Also, the presence of acriflavine and caffeine in the nutrient agar decreases the number of survivors (in common with far-u.v.).     

PHOTOPRODUCTS OF BROMOURACIL-LABELED DNA AND THE STRUCTURE OF 5-BROMODEOXYURIDYLYL-(3' → 5')-THYMIDINE PHOTOPRODUCT

Abstract— –An attempt was made to identify some of the ultraviolet (u.v.) photoproducts of 5-bromouracil-labeled DNA (BrU-DNA). Two synthetic dinucleotides, 5-bromodeoxyuridylyl-(3' →5 ')-thymidine (BrdUpT) and 5-bromodeoxyuridylyl-(3' → 5')-deoxycytidine (BrdUpdC), were prepared. Each gave a single u.v. photoproduct which in turn gave a single acid hydrolysis product. 2-¹⁴C-BrU-DNA. prepared from E. coli B3, was irradiated (275–280 nm), hydrolyzed, and paper chromatographed in four systems. Comparison with the two synthetic photoproducts showed that if present at all, BrdUpT and BrdUpdC photoproducts could account for no more than 10 and 3.5 per cent respectively of the total photoproducts. At 55 per cent conversion of BrU into photoproducts, the major ¹⁴C-photoproduct was uracil (78 per cent); the remaining 22 per cent was made up of at least six products, three of which were reversed by 232 nm irradiation.
The debrominated cyclobutane structure proposed by Haug for BrdUpT photoproduct has been shown to be incorrect. It was found to contain one atom of bromine per molecule. On the basis of nuclear magnetic resonance and u.v. spectra, two possible structures are proposed for the photoproduct, each containing an eight-membered ring.     

Damage to UV-Sensitive Cells by Short UV in Photographic Flashes

Abstract— Light emitted by electronic photographic flash units is shown to damage bacteria and human skin fibroblasts deficient in repair systems, with survival curves very similar to those produced by 254 nm short UV. The lesions induced by these flashes are as photorepairable by the photolyase enzyme as those induced by 254 nm UV and result in equivalent survival rates. Biological dosimetry performed with microorganisms highly sensitive to UV ( Escherichia coli K12 AB2480, deficient in excision and recombinational-dependent repair systems and Bacillus subtilis UVSSP spores, deficient in excision and in a specific spore repair process) revealed that each 1 ms flash of light from the photographic unit used in this work contained the equivalent of 0.25 J m⁻² of 254 nm UV, when measured at a distance of 7.0 cm. This dose of UV was found to be lethal to both repair-deficient E. coli bacteria and repair-deficient human skin fibroblasts obtained from xeroderma pigmentosum donors, as well as mutagenic in B/r wild-type and HCR-mutant bacteria.