Induction of multilocus lesions by UVC-radiation in mouse L5178Y lymphoblasts

The survival, the mutant frequency and the nature of the DNA alteration responsible for the inactivation of the thymidine kinase (tk) locus were investigated in 5 strains of mouse L5178Y lymphoblasts exposed to UVC radiation. The nature of the DNA alteration was investigated in independent TK-/- mutants using Southern blot analysis. The concomitant loss of galactokinase (GK) activity in homogenates of individual TK-/- mutants was taken as an indication that the lesion inactivating the tk allele extended to the neighboring galactokinase (gk) allele. The survival of strains LY-R16 and LY-R83 was decreased to a greater extent than that of strains LY-S1, LY-SR1, and LY-3.7.2C, reflecting a deficiency in excision repair in strains derived from LY-R cells. The TK-/- mutant frequency of strain LY-R83, which is monosomic for chromosome 11 and thus hemizygous for the tk and gk genes, was only 50% of the mutant frequency of strain LY-R16 which is heterozygous for the tk gene. Moreover, a greatly reduced percentage of individual spontaneous and UVC-induced TK-/- mutants of strain LY-R83 showed loss of GK activity in comparison to the other strains. This result indicates that UVC irradiation induces intergenic mutations and that such mutants are poorly recovered in the hemizygous strain. Strain LY-3.7.2C appears to have only one active galactokinase (gk) allele, and very few TK-/- mutants of this strain showed loss of GK activity, possibly because this strain, although heterozyogous for the tk gene, is hemizygous in the region of the gk gene. Strains LY-R16 and LY-S1 are deficient in the repair of UVC- and X-radiation-induced damage, respectively, and the percentage of TK-/- mutants with intergenic mutations was higher for strain LY-R16 after UVC-radiation and for strain LY-S1 after X-radiation. These results indicate that unrepaired DNA lesions lead to an increase in intergenic mutations.     

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radiation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

The repair of potentially lethal damage and sublethal damage in strains of mouse L5178Y lymphoma cells differing in radiation sensitivity

Mouse lymphoma strains L5178Y-R (LY-R) and L5178Y-S (LY-S), which are differentially sensitive to the cytotoxic effects of ionizing radiation, were found to differ in their abilities to repair potentially lethal damage (PLD) and sublethal damage (SLD). The results showed that strain LY-R was more proficient than strain LY-S in the repair of SLD. The split dose recovery observed in strain LY-S could be accounted for by its recovery during postirradiation incubation. In contrast, SLD repair occurred in the absence of PLD repair in strain LY-R. The possibility that the repair of PLD might be completed prior to the postirradiation incubation in strain LY-R was suggested by the decreased survival observed when the cells were irradiated in a hypotonic solution. The repair of PLD and SLD in strain LY-S was temperature sensitive, occurring during postirradiation incubations between 15 and 34 degrees C, but not at 37 or 40 degrees C. This temperature sensitivity is very similar to the temperature sensitivity of the repair of pH 9.6-labile lesions in DNA in strain LY-S, as reported previously. Thus postirradiation cellular recovery processes in strain LY-S may involve the repair of pH 9.6-labile lesions in DNA. Temperature-dependent changes in the postirradiation distribution of cells throughout the cell cycle were observed which could contribute to the temperature sensitivity of the postirradiation recovery of strain LY-S.     

Discrepancy between the initial DNA damage and cell survival after camptothecin treatment in two murine lymphoma L5178Y sublines

Two L5178Y (LY) murine lymphoma cell sublines, LY-R, resistant, and LY-S, sensitive, to X-irradiation display inverse cross-sensitivity to camptothecin (CPT): LY-R cells were more susceptible to this specific topoisomerase I inhibitor than LY-S cells. After 1 h incubation with CPT, the doses that inhibited growth by 50 per cent (ID₅₀) after 48 h of incubation were 0·54μM for LY-R cells and 1·25 μM for LY-S cells. Initial numbers of DNA–protein crosslinks (DPCs) measured at this level of growth inhibition were two-fold higher in LY-R (5·6 Gray-equivalents) than in LY-S cells (3·1 Gray-equivalents), which corresponds well with the greater in vitro sensitivity of Topo I from LY-R cells to CPT.^1,2 Conversely, the initial levels of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) were lower in LY-R cells (4·2 Gray-equivalent SSBs and 5·8 Gray equivalent DSBs) than in LY-S cells (8·0 Gray-equivalent SSBs and 12·0 Gray-equivalent DSBs). Dissimilarity in the replication-dependent DNA damage observed after 1 h of treatment with CPT was not due to a difference in the rate of DNA synthesis between the two cell lines, but may have arisen from a substantially slower repair of DNA breaks in LY-S cells.³ Release from G₂ block by caffeine co-treatment significantly increased cell killing in the LY-S subline, and only slightly inhibited growth of LY-R cells. These results show that after CPT treatment cells arrest in G₂, allowing them time to repair the long-lived DSBs. As LY-S cells are slower in repairing the DSBs, they were more susceptible to CPT in the presence of caffeine.     

The influence of dose rate on the lethal and mutagenic effects of X-rays in proliferating L5178Y cells differing in radiation sensitivity

The lethal and mutagenic effects of ionizing radiation delivered at high (53 Gy/h) and low (0.02 Gy/h) dose rates were measured in two closely related strains of mouse lymphoma L5178Y cells differing in radiation sensitivity (LY-R and LY-S). Strain LY-R was more resistant to the lethal effects of radiation than strain LY-S when exposed at either the high or low dose rate. The survival of strain LY-R was markedly enhanced by the reduction in dose rate. The dose-rate dependence of the survival of strain LY-S was less clear, because of the biphasic nature of its survival curve following low dose-rate radiation. However, if the initial slope of the low dose-rate survival curve is compared to the slope of the high dose-rate survival curve for strain LY-S, only a slight increase in survival at the low dose rate is apparent. Although more sensitive to the lethal effects of radiation, strain LY-S was less mutable at the hypoxanthine/guanine phosphoribosyl transferase locus by both low dose-rate and high dose-rate radiation than strain LY-R. Little dose-rate dependence was exhibited by either strain with regard to the mutagenic effects of radiation. Thus, for strain LY-R, which showed marked dose-rate dependence for survival but not for mutation, the ratio of mutational to lethal lesions was much greater following exposure to low dose-rate than to high dose-rate radiation.     

Repair of gamma-ray-induced base damage in L5178Y sublines is damage type-dependent and unrelated to radiation sensitivity.

The L5178Y (LY) murine lymphoma sublines LY-R and LY-S are differentially sensitive to ionizing radiation. The high radiation sensitivity of LY-S cells is related to impaired rejoining of DNA double strand breaks. We found previously that the gamma-ray-induced base damage is higher in the more radiosensitive LY-S subline. Here, we examine the role of the repair of ionizing radiation induced base damage in relation to the radiosensitivity difference of these sublines. We used the GS/MS technique to estimate the repair rates of six types of base damage in gamma-irradiated LY cells. All modified DNA bases identified in the course of this study were typical for irradiated chromatin. The total amount of initial base damage was higher in the radiation sensitive LY-S subline than in the radiation resistant LY-R subline. The repair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were similar in both cell lines, the repair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the repair of 5-OHUra was faster in its radioresistant counter, the LY-R. Altogether, the repair rates of the y-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the differential lethal or mutagenic effects of ionizing radiation in these sublines.     

Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells

In previous studies, the two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. Thus, in comparison to strain LY-R, strain LY-S has been found to be more sensitive to the lethal effects of ionizing radiation, more resistant to the lethal effects of UV radiation, but less mutable at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus by both UV and X-radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na+/K+-ATPase locus as well as the HGPRT locus was determined. As previously reported, we found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, we found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na+/K+-ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na+/K+-ATPase locus. We have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells.     

DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells

DNA supercoiling ability was assayed following irradiation in two cell lines of differing radiosensitivity, L5178Y-S (LY-S) and L5178Y-R (LY-R). Cells treated with NaCl and Triton X-100 were exposed to increasing concentrations of the fluorescent, DNA-intercalating dye, propidium iodide (PI), and the diameter of the resulting fluorescent halo of DNA was measured. As the PI concentration was increased from 0.5 to 5 micrograms/ml, halo diameter increased from 20-25 to 45-55 microns due to the unwinding of the DNA supercoils. This process was similar for both cell lines under all conditions studied. As the PI concentration was increased to 50 micrograms/ml, the halo rewound to a diameter of 25-30 microns in unirradiated cells from both lines. However, following exposure to 3-12 Gy of 137Cs gamma rays, the ability of the DNA to be rewound was inhibited in a dose-dependent manner. Rewinding inhibition was greater in LY-S cells than in LY-R cells. Since the induction of DNA damage (e.g., single-strand DNA breaks) appears to be the same for both cell lines, this result implies that a similar extent of damage results in a greater loss of topological constraints on the DNA loops in LY-S. Such a change might be related to the protein composition of the nucleoid cores. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that nucleoids from LY-S cells were missing a 55-kDa protein present in LY-R.     

DNA repair,cell cycle progression and cell death following camptothecin treatment in two murine lymphoma L5178Y sublines

The processes involved in cell response to camptothecin (CPT) were investigated in two sublines of L5178Y (LY) murine lymphoma; LY-R, resistant and LY-S, sensitive to X-irradiation, which are inversely cross-sensitive to the drug. The cells were pulse-treated with 2 μM CPT for 1 h; this resulted in equal numbers of replication-related DNA double-strand breaks (DSBs) in both sublines.¹ After drug removal, at different time points up to 24 h, the levels of DSBs were measured by using field inversion gel electrophoresis (FIGE) and comet assay at neutral pH. Both methods revealed faster DSBs repair in LY-S than in LY-R cells, in contrast with X-ray-induced DSBs. This however, was followed by the appearance of secondary breaks in the former subline. The cell cycle arrest was at S/G₂ phase and comprised equal numbers of cells in LY-S and LY-R populations. In both sublines formation of giant cells took place, as well as delayed apoptosis starting about 20 h post-CPT incubation and proceeding with similar intensity. At the same time, the total number of necrotic cells appearing during post-exposure incubation in the LY-R subline exceeded that in the LY-S subline. We suggest that, beside previously documented higher susceptibility of topoisomerase I (Topo I) from LY-R cells to CPT,^2,3 a higher initial rate of replication-related DSBs repair, but not lower propensity to apoptosis, may contribute to the relative CPT resistance of LY-S versus LY-R cells. Copyright © 1998 John Wiley & Sons, Ltd.     

Novobiocin treatment reverses radiation-induced alterations in higher-order DNA structure in L5178Y nucleoids

We have studied the effect of novobiocin treatment on radiation-induced damage and its repair in higher-order DNA structure in two mouse leukemia cell lines differing in their radiosensitivity, L5178Y-R (LY-R) and L5178Y-S (LY-S). We used the fluorescent halo technique to measure alterations in the superhelical density and the topological constraints of DNA in LY-R and LY-S nucleoids. The results for untreated cells show that both cell lines reached maximal DNA unwinding at the same concentration of propidium iodide (PI), whereas LY-S nucleoids were less efficient in their ability to rewind their DNA. The loop size did not differ significantly between the cell lines. Incubation of LY-R and LY-S cells with novobiocin at a concentration which does not influence survival (0.1 mM for 45 min), but inhibits DNA synthesis in LY-R cells (by 28%) to a greater extent than in LY-S cells (by 10%), also causes more DNA unwinding in LY-R nucleoids than in LY-S nucleoids. However, a decreased superhelical density was observed in nucleoids from both cell lines. Novobiocin applied before, and present during, irradiation prevents radiation-induced alterations in DNA supercoiling more efficiently in LY-R than in LY-S cells. The presence of novobiocin during the repair period increased DNA rewinding to levels not significantly different from control values in nucleoids from both cell lines.     

Differential antimutagenicity of WR-1065 added after irradiation in L5178Y cell lines

The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.     

Effects of low dose rate (0.003-0.025 Gy/h) chronic X-irradiation on radioresistant and radiosensitive L5178Y mouse lymphoma cells

Cultures of radioresistant (LY-R) and radiosensitive (LY-S) strains of L5178Y mouse lymphoma cells were exposed continuously to X-rays delivered at dose rates ranging from 0.003 to 0.025 Gy/h for up to 35 days. Populations of both strains proliferated actively during the exposure, but the growth rates were reduced in a dose rate-dependent manner. The reduction of growth rate occurred for strain LY-S earlier during the exposure and at lower dose rates than for strain LY-R. The survival (as measured by colony forming ability) of strain LY-R was affected only slightly at all dose rates applied. For strain LY-S, a decrease in the surviving fraction was observed in the initial part of the exposure. This decrease was followed by a plateau and eventually by an increase, in some cases to values close to the control level. The increase in the surviving fraction indicated that the radioresistance of the exposed LY-S cells had increased. This pattern was particularly clear for dose rates greater than 0.014 Gy/h. The pre-irradiated cells exhibited radioresistance when exposed to acute X-radiation after termination of the chronic exposure. The increase in radiation resistance was stable for at least 70 days after termination of the protracted exposure. These results show that mutagenic and/or selective phenomena leading to an increase in radiation resistance of mammalian cells can be caused by protracted exposures to X-rays at dose rates permitting active proliferation.     

The response of L5178Y lymphoma sublines to oxidative stress: antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-kappaB

We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.     

Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).     

Comparison of the Escherichia coli umu+-encoded function with plasmid R46-mediated error-prone repair in DNA-damaged cells

Escherichia coli strain TK701 umu+ was more resistant than strain TK702 umu when tested against bleomycin (BLM), cis-platinum(II) diamminodichloride (PDD), ultraviolet light and methyl methanesulphonate (MMS), which produce single-strand DNA damage. However, the umu mutant was no more sensitive to mitomycin C (MTC) or proflavine (PF), which cause double-strand DNA binding. Strain TK702 umu was nonmutable by any of the agents, whereas mutations were induced in the wild-type strain by PDD, UV, MMS and MTC. The E. coli umu+ function therefore mimics plasmid R46-mediated error-prone repair in protecting only against single-strand DNA damage, whilst enhancing mutagenesis by both single- and double-strand damaging agents. Comparison of plasmid R46-mediated protection and mutagenesis in umu+ and umu strains indicated that the plasmid confers a greater error-prone DNA-repair activity in the mutant. Results are discussed in terms of analogy between host umu+ and plasmid muc+ functions.     

Detection of genetic alterations induced by low-dose X rays: analysis of loss of heterozygosity for TK mutation in human lymphoblastoid cells

To elucidate the genetic influence of low-dose ionizing radiation at the chromosome level, we exposed human lymphoblastoid TK6-20C cells to 10 cGy of X rays. The TK mutation frequency was 5.7 +/- 1.3 x 10(-6) at the background level and 6.9 +/- 2.8 x 10(-6) after X irradiation. Although this small increase was not statistically significant (P = 0.40), we applied multilocus analysis using 4 TK locus markers and 12 microsatellite loci spanning chromosome 17 for TK mutants exhibiting loss of heterozygosity (LOH). The analysis demonstrated a clear effect of low-dose ionizing radiation. We observed radiation-specific patterns in the extent of hemizygous LOH in 14 TK mutants among the 92 mutants analyzed. The deleted regions in these patterns were larger than they were in the control mutants, where those restricted to the TK locus. Surprisingly, the radiation-specific LOH patterns were not observed among the 110 nonirradiated TK mutants in this study. They were identified previously in TK6 cells exposed to 2 Gy of X rays. We consider these hemizygous LOH mutants to be a result of end-joining repair of X-ray-induced DNA double-strand breaks.     

Modulation of the effect of camptothecin in x-irradiated L5178Y-R and L5178Y-S cells by benzamide

The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 μM CPT enhanced the radiosensitivity of LY-S cells (D₀ decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly(ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D₀ decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e. g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases α and δ) applied concomitantly with CPT in cells not pre-treated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication. Received: 28 November 1995 / Accepted in revised form: 20 April 1996     

Separate Branches of the uvr Gene-Dependent Excision Repair Process in Ultraviolet-Irradiated Escherichia coli K-12 Cells; Their Dependence upon Growth Medium and the polA, recA, recB, and exrA Genes

The extent of repair of single-strand breaks (incision breaks) induced in the deoxyribonucleic acid (DNA) of Escherichia coli K-12 cells by the uvr gene-dependent excision repair process after ultraviolet (UV) radiation was determined in the wild-type, polA1, recA56, recB21, and exrA strains. The wild-type strain repaired all incision breaks after incident doses of UV radiation (254 nm) of approximately 60 J m(-2) or less when incubated in growth medium, or approximately 15 J m(-2) or less when incubated in buffer. The polA1 strain repaired the incision breaks completely after incident doses of approximately 12 J m(-2) or less when incubated in growth medium, or after approximately 4 J m(-2) when incubated in buffer. The recA13, recB21, and exrA strains showed essentially complete repair after incident doses of 10 to 15 J m(-2) whether the cells were incubated in buffer or growth medium. These results suggest that the uvr gene-dependent excision repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec(+)exr(+) genotype, and a second which can occur in buffer (growth medium-independent) and is largely dependent on DNA polymerase I. The presence of chloramphenicol in the growth medium resulted in an inhibition of the growth medium-dependent repair occurring in wild-type and polA1 cells and had little or no effect on the extent of repair observed in recA56, recB21, or exrA cells. The similarities between the growth medium-dependent and -independent branches of excision repair and two known processes for the repair of X-ray-induced single-strand breaks are discussed.     

Survival and the repair of single-stranded DNA breaks in gamma-irradiated Escherichia coli cells adapted to methylmethane sulfonate]

The survival and repair of single-strand breaks of DNA in gamma-ray-irradiated E. coli adapted to MMS (20 mkg/ml during 3 hours) have been investigated. It is shown that the survival of adapted bacteria of radioresistant strains B/r, H/r30, AB1157 and W3110 pol+ increases with DMF (dose modification factor) ranging within 1.4-1.8 and in radiosensitive strains Bs-1, AB1157 recA13 and AB1157 lexA3 with DMF ranging within 1.3-1.4, and does not change in strains with mutation in polA gene P3478 polA1 and 016 res-3. There is no increase in radioresistance during the adaptation to MMS under the action of the protein synthesis inhibitor chloramphenicol. The increase in radioresistance during the adaptation to MMS correlates with the acceleration of repair of gamma-ray-induced single-strand breaks in the radioresistant strains B/r and W3110 pol+ and with the appearance of the ability to repair some part of DNA single-strand breaks in the mutant Bs-1, which beyond the adaptation to MMS does not repair these damages. The incomplete reparability of DNA single-strand breaks in P3478 polA1 strain cells, both adapted and non-adapted to MMS, is equal.