Dark Repair Processes Research Articles

The variation in UV sensitivity of four K 12 strains of Escherichia coli as a function of their stage of growth

The UV response of four K12 strains of E. coli that differ in their repair characteristics was studied as a function of their stage of growth. The four strains used were AB1157 that possesses the full complement of repair genes, the excision-deficient AB-1886 ( uvr A-6), the recombination-deficient AB2463 ( recA-13) and the excision- and recombination-deficient AB2480 ( recA-13, uvrA-6). The UV sensitivity-growth phase profile of the double mutant showed a decrease in early exponential phase and then an increase to the original level by the start of stationary phase that remained unchanged up to 48 h. This decreased sensitivity was found to be associated with an increase in the shoulder of the full UV survivor curve. Deviations from the AB2480 profile shown by other strains reflect the influence of repair mechanisms on the UV-induced damage in cells, and indicate the influence that physiological changes occurring during different phases of the growth cycle have on the efficiencies of the repair systems. Since AB1886 showed a similar profile to AB2480, it appears that postreplicatory recombination repair is unaffected by the intracellular changes. However, the recombination-negative AB2463 showed a marked increase in UV sensitivity throughout exponential growth followed by an increase in resistance that continued up to 48 h. It is postulated that this increased sensitivity in rapidly dividing cells results from a decreased repair efficiency due to a decrease in time before DNA replication takes place and also to a temporary decrease in numbers of repair enzyme molecules within the cells. The enzyme concentration increases in stationary phase and accounts for the enhanced recovery noted during this stage of growth. Results of experiments on pre-irradiation and postirradiation liquid holding of cells support this view. Studies with AB1157 indicate the interrelationship of excision and recombination, working sequentially, at different stages of the growth cycle. A comparison of the AB1157 results with those from single mutants indicate that while both processes are necessary for maximum cellular recovery, in most cases recombination can compensate for decreased excision repair efficiency. However, when excision efficiency falls below a certain limit, recombination can no longer fully cope with the partially repaired cells and an increased sensitivity results. Possible reasons for the interaction of these two dark repair processes are discussed.

X-irradiation sensitivity in Escherichia coli defective in DNA replication.

A mutant of Escherichia coli with a temperature sensitive defect in DNA replication is sensitive to X-irradiation but not to UV-irradiation. After UV-irradiation, dark-repair processes—dimer excision, DNA breakdown, repair synthesis and DNA strand joining—appear normal at the restrictive temperature. After X-irradiation, DNA degradation exceeds that in the wild type, and irradiation-dependent DNA synthesis does not occur. Single-strand breaks introduced into the DNA by the irradiation are nor repaired. The data indicate that the mutation results in a defect in repair of X-ray induced single-strand breaks as well as a defect in DNA replication. They provide evidence for the existence of a repair pathway for X-irradiated DNA similar to, but at least partially independent from, that postulated for the dark-repair of UV-irradiated DNA, viz., degradation at the site of the lesion, resynthesis of the degraded DNA complement and ligation of the DNA strand.

Ribosome degradation in ultraviolet light-damaged bacteria starved for an amino acid

Degradation of RNA and of 30-S and 50-S ribosomes occurred when Escherichia coli H/r 30R cells were exposed to 400 ergs/mm 2 of ultraviolet light and then incubated without arginine, a required amino acid. Under these conditions, the ribosomes remained stable for 1 h, but rapid breakdown began thereafter and eliminated the greater part of the ribosomes by 2 h. Arginine prevented most of the breakdown. Both the photoreversible pyrimidine dimer and non-photoreversible damage appeared to result in breakdown, but at this ultraviolet light dose most of the damage was not photoreversible. If 30 min of postirradiation incubation with arginine was allowed prior to incubation without the amino acid, little degradation occurred on subsequent arginineless incubation, indicating that dark repair processes dependent on protein synthesis are capable of removing the cause of the breakdown, presumably through repair of damage to DNA resulting in recovery of capacity for DNA replication.

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.

Actinomycin D inhibition of UV-dark repair in [formula omitted][formula omitted

Actinomycin D inhibits dark repair of UV-damage in Bacillus subtilis . This inhibition is not due to an inhibition of mRNA or induced enzyme synthesis. Actinomycin D prevents the removal of UV photoproducts from DNA. After a lag, breakdown of DNA occurs in UV irradiated cells in the presence of actinomycin. It appears likely that there is no direct relationship between the dark repair process and the massive breakdown of DNA to acid-soluble fragments.

Repair mechanisms and variations in UV sensitivity within the cell cycle

1. 1. Studies were made of the UV sensitivity of a series of stages in the cell cycle of synchronous populations of diploid meiotic spores of Chlamydomonas reinhardi. 2. 2. Early stages had sigmoid survival curves, but at successively later stages of development, D Q values were continuously reduced, and the latest stages had exponential survival curves. D O values, though changing only to a minor extent, were at a maximum in mid-cycle. 3. 3. From studies of the effects of various post-irradiation treatments and in particular of the effect of inhibitors of dark-repair processes, it was concluded that much of the variation in stage sensitivity was attributable to different repair activities. Those stages farthest from division had the longest period in which to repair UV lesions, whereas successively later stages had less time and thus exhibited an enhanced sensitivity.

The Induction of Thymine Dimers in Ultraviolet-Irradiated Mammalian Cells

It is well established that both gene mutations and chromosome aberrations can be induced by ultraviolet (UV) light (see review: 1). The action spectrum for mutation implicates nucleic acid absorption (2), and the action spectrum for the UVinduced chromosome aberrations in Tradescantia pollen grains approximately parallels the absorption spectrum of nucleic acids (3). Similarly, the frequency of UVinduced chromosome aberrations in mammalian cells varies with wavelength and reaches a peak around 2650 A (4). It has been further demonstrated that substitution of thymidine by 5-bromodeoxyuridine or 5-iododeoxyuridine greatly enhances the UV-sensitivity in the induction of mammalian chromosome breaks. These results suggest that DNA is the primary target of the UV injury that leads to chromosome aberrations (4). The effects of UV on nucleic acids and their constituents have been the subject of numerous investigations (see reviews: 5, 6). The kinds of UV damage to DNA include photohydration, cross-linkage, and the formation of thymine dimer. In particular, recent work has indicated that thymine dimerization may account for a large proportion of the UV-induced damage to transforming activity (7) and probably for much of the loss of DNA synthetic capacity in bacteria (8, 9). It has been found that the photoreactivation of transforming principle can be accounted for by dimer splitting (7). Furthermore, in certain strains of Escherichia coli, UV-induced thymine dimers, together with a few neighboring nucleotides, can be excised by what seems to be a dark repair process (10, 11). If the conclusion is correct that chromosomal DNA is the site of UV injury that leads to chromosome breaks, it is of interest to determine the nature of the induced molecular alteration(s) responsible for chromosome breaks. This report presents