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R M Tyrrell

Publications and source records attributed to R M Tyrrell.

At least 91 records · Page 5Linked to original sources
PubMed1985

Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: evidence that alpha- and beta-polymerases act sequentially at the repolymerisation step.

We have characterised far-ultraviolet-radiation-induced DNA-repair synthesis in permeabilised arrested (non-dividing) primary human skin fibroblasts. Approximately half the maximum repair synthesis is seen after a UV fluence of 4.0 Jm-2 and little additional incorporation was observed at fluences above 20.0 Jm-2. UV-damaged permeable cells were treated with specific inhibitors of DNA polymerase alpha and beta, both alone and in combination. The degree of inhibition of repair incorporation by aphidicolin indicates that polymerase alpha is involved in the majority (85-90%) of repair synthesis after both high and low (less than 4.0 Jm-2) UV fluences. Dideoxythymidine triphosphate seems able to inhibit DNA-repair synthesis only when polymerase alpha is fully or almost fully functional, indicating that polymerase beta is unable to substitute in repair for an alpha polymerase blocked by aphidicolin. These data suggest that the two enzymes may act sequentially to complete repair patches rather than acting independently.

Cells, Cultured↗
PubMed1985

Excision repair in u.v. (254 nm) damaged non-dividing human skin fibroblasts: a major biological role for DNA polymerase alpha.

We have used the eukaryotic DNA polymerase alpha inhibitor, aphidicolin, and the polymerase beta inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (less than 3 J m-2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 micrograms/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase beta inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the beta polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase alpha appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.

Aphidicolin↗
PubMed1985

Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision step.

The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested (nondividing) human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 micrograms ml-1 nalidixic acid and 1.0 micrograms ml-1 aphidicolin is unchanged when compared with that due to treatment with 1.0 micrograms ml-1 aphidicolin alone, while that for 150 micrograms ml-1 novobiocin + 1.0 micrograms ml-1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 micrograms ml-1 novobiocin inhibited repair synthesis by approximately 60% over the fluence range employed. Nalidixic acid at a concentration of 300 micrograms ml-1 caused no detectable inhibition of repair synthesis. We conclude that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage.

Aphidicolin↗
PubMed1984

Mutagenic action of monochromatic UV radiation in the solar range on human cells.

Mutations to ouabain resistance (selecting for base modifications at the co-dominant Na+K+-dependent ATP-ase locus) and thioguanine resistance (selecting for a wide range of genetic changes at the recessive hypoxanthine-guanine phosphoribosyl transferase locus) were measured in a repair-proficient human lymphoblastoid line with defined monochromatic radiations in the UVC (254 nm), UVB (302 nm, 313 nm), UVA (334 nm, 365 nm) and visible (405 nm) ranges. No mutations were detected at wavelengths in the range 334-405 nm. At 254 nm and 313 nm, both mutations to thioguanine resistance and survival were consistent with those expected from the relative levels of cyclobutane-type pyrimidine dimers induced. However, at 313 nm, the ratio of ouabain-resistant to thioguanine-resistant mutants is 10 times higher than at 254 nm, indicating that a unique type of pre-mutagenic base damage is induced at the longer wavelength. Radiation in the UVA (334 nm) range reduced the induction of mutations by a UVC (254 nm) wavelength at both mutation markers. These results suggest, first, that distinct types of biologically expressed genetic damage may be induced in the UVB region of sunlight and, second, that strong interactions may occur between the different wavelength regions of sunlight that can modify the expression of this genetic damage in human cells.

Cell Line↗
PubMed1984

Lack of induction of non-targeted mutations in intact bacteriophage by UVB (313 nm), UVA (334 nm, 365 nm) and visible (405 nm) irradiation of host cells.

Mutation to virulence has been measured in intact bacteriophage lambda 15 infected into host cells pre-treated with UVC (254 nm), UVB (313 nm), UVA (334 nm, 365 nm) or visible (405 nm) radiations. We have confirmed that UVC radiation leads to a large enhancement (maximum enhancement factor of 140 in wild-type) of the background spontaneous mutation frequency (non-targeted mutagenesis) and have further shown that this is at least partially dependent on excision repair (maximum enhancement factor of 14 in uvrA strain). In contrast, UVB (313 nm) radiation enhances the mutation frequency by less than a factor of 2. Longer wavelength UVA radiation (334 nm, 365 nm) actually reduces the mutation frequency to 25% of the background levels presumably by reducing the levels of viral replication occurring in the host cells. A visible wavelength (405 nm) has no effect on mutation frequency over the fluence range employed.

Bacteriophage lambda↗
PubMed1984

Exposure of nondividing populations of primary human fibroblasts to UV (254 nm) radiation induces a transient enhancement in capacity to repair potentially lethal cellular damage.

Nondividing (arrested) populations of primary human fibroblasts from normal individuals exposed to an initial dose (1.5 or 3 J X m-2) of far-UV (254 nm) radiation and then incubated in medium containing low (0.5%) serum develop enhanced resistance to inactivation of cloning efficiency by a second (challenge) dose of UV. The resistance develops within 2-4 days, after which there is a decline. Resistance develops to a higher degree and more rapidly (1-2 days) in cells derived from patients with the variant form of xeroderma pigmentosum. Excision-deficient cells from xeroderma pigmentosum complementation group A individuals also develop UV resistance after a lower (0.2 J X m-2) exposure to UV. Enhanced UV resistance does not develop in UV-irradiated cell populations incubated with the protein synthesis inhibitor cycloheximide (5 microM). These observations are consistent with the interpretation that exposure of human fibroblasts to low doses of UV induces synthesis of a protein involved in a metabolic pathway that transiently enhances the capacity of cells to repair potentially lethal damage resulting from a subsequent dose of UV.

Cell Division↗
PubMed1983

Specific toxicity of aphidicolin to ultraviolet-irradiated excision proficient human skin fibroblasts.

Aphidicolin, a specific inhibitor of the eucaryotic alpha polymerase, has been employed to study the role of this enzyme in repair of potentially lethal damage (PLD) induced by far u.v. (254 nm) radiation in normal and repair defective primary human fibroblasts. There is strong concentration dependent specific toxicity to cells treated with a fluence of 6 Jm-2 of far-u.v. radiation and incubated with aphidicolin for 2 days over the concentration range 0.0025-2.5 micrograms/ml. A similar effect is seen with a xeroderma pigmentosum (XP) variant (excision proficient) strain but there is no specific toxicity to u.v. irradiated excision deficient XP cells of complementation group A. Inactivation of irradiated excision proficient fibroblasts is rapid over the first 6 h of aphidicolin (1 microgram/ml) treatment but the reaction takes 2 days or longer to complete depending on the u.v. dose. These results demonstrate that the apparent uncoupling of excision repair seen previously by other investigators prevents repair of PLD and is lethal to the cells.

Antibiotics, Antineoplastic↗
PubMed1983

Induction of oxygen-dependent lethal damage by monochromatic UVB (313 nm) radiation: strand breakage, repair and cell death.

The action of 313 nm radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair proficient strain and three repair deficient strains (polA, recA, uvrA) of Escherichia coli K-12. Although the induction of breaks was linear in purified T4DNA (6.3 X 10(-4) breaks/2.5 X 10(9) daltons/Jm-2) and the polA strain (4 X 10(-4) breaks/2.5 X 10(9) daltons/Jm-2), simultaneous induction and repair of breaks were observed in the uvrA, recA and repair proficient strains at doses less than 5 X 10(4) Jm-2. The final rates of induction in these strains were 1 X 10(-4), 7.5 X 10(-5) and 7.5 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively. A highly efficient polA-dependent repair occurring at 0 degree C in minimal buffer and a second slower type of repair occurring at 31 degrees C in the polA strain were detected. Oxygen dependence of cellular inactivation was observed for the polA and repair proficient strains irradiated at 313 nm thus providing biological evidence for an oxygen-dependent lesion involved in lethality in the short wavelength range of the solar u.v. The lower hypoxic break induction rates of the polA (1.6 X 10(-4) breaks/2.5 X 10(9) daltons/Jm-2) and the repair proficient (3.6 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2) strains, indicate oxygen-enhanced DNA breakage by 313 nm radiation.

Aerobiosis↗
PubMed1981

Mutagenic interactions between near-ultraviolet (365 nm) radiation and alkylating agents in Escherichia coli.

The mutagenic interaction between near-ultraviolet (365 nm) radiation and the alkylating agents ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS) was studied in a repair-competent and an excision-deficient strain of Escherichia coli. Near-UV radiation modified the metabolic response of exposure to these chemicals and either reduced or increased their mutagenic efficiency. Based on these results, an experimental model was formulated to explain the mutagenic interactions that occur between near-UV and various agents that induce prototrophic revertants via error-prone repair of DNA. According to this model, low doses of near-UV provoke conditions for mutation frequency decline (MFD) and lead to a mutagenic antagonism. With increasing near-UV doses, damage to constitutive error-free repair systems increases, favouring the error-prone system and inhibiting the MFD. Under these conditions there will be a progressive decrease in antagonism until at high doses an enhancement of mutation frequency (positive interaction) will occur.

DNA Repair↗
PubMed1981

Excision repair of aflatoxin B1-DNA adducts in human fibroblasts.

The processing of covalent aflatoxin B1 (AFB1)-DNA adducts was investigated in confluent normal fibroblasts (NF) and xeroderma pigmentosum skin fibroblasts of Complementation Group A (XPA) following treatment with rat liver microsome-activated AFB1 for 30 min. Following rapid DNA isolation at slightly acidic pH by a new filter technique, more than 90% of the adducts corresponded to 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-AFB1 (AFB1-N7-Gua) according to the analysis of acid hydrolysates by high-pressure liquid chromatography. The changes in adduct concentration and composition were compared between DNA isolated immediately following AFB1 treatment and incubated at neutrality in vitro and DNA in situ in the cell isolated after different lengths of incubation. The following conclusions were reached from the observed differences in the kinetics of adduct removal from free DNA and DNA in situ in NF and XPA: (a) AFB1-N7-Gua is removed spontaneously and enzymatically in NF but probably only spontaneously in XPA. This result suggests that these lesions are removed via nucleotide excision repair in NF; (b) the putative 2,3-dihydro-2(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 is formed in a secondary reaction from AFB1-N7-Gua in vitro and in situ in the cell. It accumulates more rapidly and to a greater extent in XPA than in NF and persists in both cells types over prolonged periods. The reaction of AFB1-N7-Gua to 2,3-dihydro-2-(N5-formyl-2',5'6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatox in B1 represents the transformation of a repairable lesion to a nonrepairable, persistent lesion.

Aflatoxin B1↗