Quantitative measurement of DNA strand breaks and repair in gamma-irradiated human leukocytes from normal and ataxia telangiectasia donors

Fluorimetric analysis of DNA unwinding, which allows measurement of DNA strand breaks in human leukocytes, has been optimized by reducing the amount of cells required for the test and by modifying the DNA alkali unwinding conditions. This permitted measurement of DNA strand-break induction in cells irradiated with low (0.5-7 Gy) or high doses (5-20 Gy) of gamma rays. Linear dose-response curves were obtained for both dose ranges. Presence of cysteamine during irradiation caused a decrease in the extent of DNA strand breaks. The kinetics of the DNA strand-break rejoining process appeared to be biphasic over the dose range of 2-20 Gy when plotted on a linear vs linear axis (percentage of damage as a function of time). Since the rate of disappearance of damaged DNA was similar for any given dose and for all postirradiation incubation times tested, we have expressed the extent of repair after a given postirradiation incubation as the ratio of the slopes of the regression lines obtained from incubated and nonincubated cells. Leukocytes from 25 healthy donors were analyzed to determine an average value for controls. No difference in the level of DNA strand breaks and the rate of repair of these breaks was observed between leukocytes from three ataxia telangiectasia patients and those from normal donors.     

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organisation of repair

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the "random collision" model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a "processive" mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. These two models give different predictions concerning the time course of repair. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate of repair until scanning is complete. We have examined the time course of DNA repair in human fibroblasts given low (generally sublethal) doses of UV light. Using 3 distinct assays, we find no sign of a constant repair rate after 4 J/m2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. The implications of this finding for the structural organisation of repair are discussed.     

UV-induced repair inEscherichia coli B/r Hcr− thy trp cells: Its dependence on UV damage

Irradiation ofEscherichia coli B/r Hcr^? thy trp cells with a low UV-dose permits a post-replication repair of DNA and decreases the breakdown of DNA after a successive irradiation of cells with high UV doses. The usefulness of a repair function of the protein synthesized after a low irradiation dose increases with the increasing damage of DNA.     

Is repair of DNA strand break damage from ionizing radiation second-order rather than first-order? A simpler explanation of apparently multiexponential repair.

The purpose of this paper is to suggest the hypothesis that repair of radiation damage might be largely a second-order process (binary), as well as or instead of first-order (monoexponential). Second-order means that the rate of repair is proportional to n(2) instead of to n, where n is the number of repairable breaks. Integrating this equation gives a linear plot of the reciprocal proportion of unrepaired lesions, n(0)/n(t), as a function of repair time. This is in contrast to mono- or biexponential processes which give rise to reciprocal plots not consistent with such linearity, except with specially selected distributions with multiple T((1/2))'s. There is the advantage of only one parameter (the first half-time) instead of (2n - 1) parameters for n components. At times greater than 2tau of the longest exponential component, a larger proportion of damage would be incompletely repaired than in a mono- or biexponential model of repair. Data on DNA repair from published laboratory experiments were reanalyzed. Results are presented as graphs of the reciprocal of the proportion of damage remaining as a function of time after irradiation of DNA. If the second-order process is correct, these graphs should be straight lines, even though traditional semilog plots of the same data are markedly concave upward, showing the well-noted slowing down of repair with time after irradiation. All the data sets found in the literature showed a good fit to a straight line representing reciprocal repair. Repair of single-strand breaks in DNA fitted very well, from 1.0 down to 1/40 of the initial damage remaining, with tau values of 5-10 min. Repair of DSBs fitted almost as well. One set of data showed a strong dependence on temperature in the range 10-37 degrees C, with each curve fitting the straight reciprocal plot. The tau values for DSBs were 10-100 min, of similar magnitude to those for repair of animal tissues. The second-order process with a single time parameter could explain the data showing "apparently slowing down" repair previously analyzed by multiexponential formulae requiring more parameters. It appears that second-order repair may play a larger part in repair processes than has usually been assumed. It is suggested that analysis of data on repair of radiation-induced damage could test the second-order (one-parameter reciprocal) analysis, as well as using bi-or multiexponential analyses. If repair in DNA is relevant to recovery in mammalian tissues, there may be serious clinical implications, to be discussed elsewhere.     

Controlled degradation by ClpXP protease tunes the levels of the excision repair protein UvrA to the extent of DNA damage

UV irradiation damages DNA and activates expression of genes encoding proteins helpful for survival under DNA stress. These proteins are often deleterious in the absence of DNA damage. Here, we investigate mechanisms used to regulate the levels of DNA-repair proteins during recovery by studying control of the nucleotide excision repair (NER) protein UvrA. We show that UvrA is induced after UV irradiation and reaches maximum levels between ∼20 and 120 min post UV. During post-UV recovery, UvrA levels decrease principally as a result of ClpXP-dependent protein degradation. The rate of UvrA degradation depends on the amount of unrepaired pyrimidine dimers present; this degradation rate is initially slow shortly after UV, but increases as damage is repaired. This increase in UvrA degradation as repair progresses is also influenced by protein–protein interactions. Genetic and in vitro experiments support the conclusion that UvrA–UvrB interactions antagonize degradation. In contrast, Mfd appears to act as an enhancer of UvrA turnover. Thus, our results reveal that a complex network of interactions contribute to tuning the level of UvrA in the cell in response to the extent of DNA damage and nicely mirror findings with excision repair proteins from eukaryotes, which are controlled by proteolysis in a similar manner.     

Effect of dithiocarbamate pesticide zineb and its commercial formulation,azzurro. IV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells

Single cell gel electrophoresis (SCGE) was used to analyse dithiocarbamate zineb- and the zineb-containing technical formulation azzurro-induced DNA damage and repair in CHO cells. Cells were treated with zineb (50.0 microg/ml) or azzurro (100.0 microg/ml) for 80min, washed and reincubated in pesticide-free medium for 0-12h until SCGE. Viability of treated cells (0 h) did not differ from control remaining unchanged up to 6h of incubation. After 12h, viability decreased up to 70 and 54% in zineb- and azzurro-treated cultures, respectively. SCGE revealed at 0 h the absence of undamaged cells and an increase of slightly damaged and damaged cells in zineb-treated cultures or by an increase in damaged cells in azzurro-treated cultures. For both chemicals, a time-dependent repair of pesticide-induced DNA damage within a 0-12h post-treatment incubation period was observed. Overall, damaged cells decreased as a function of the repair time for both pesticides while the slightly damaged cells decreased as a function of the repair time of zineb-induced DNA damage. Concomitantly, a time-dependent increase of undamaged cells was observed within the 0.5-12h repair time for both pesticides. At 12h after treatment, no differences in the frequencies of undamaged, slightly damaged and damaged cells were found between both zineb- or azzurro-treated cultures and control values as well as between zineb- and azzurro-treated cells. Immediately after exposure, nuclear DNA from zineb and azzurro-treated cells were larger and wider than nuclear DNA from untreated cells. When damaged cells were allowed to repair, a time-dependent decrease of the amount of free DNA migrating fragments was observed committed only to damaged cells but not in slightly or undamaged cells. On the other hand, no time-dependent alteration on nuclear DNA width within the 0-12h repair period was observed.     

DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests

A population study is reported in which the DNA damage induced by g-radiation (2 Gy) and the kinetics of the subsequent repair were estimated by the comet and micronucleus assays in isolated lymphocytes of 82 healthy donors and patients with head and neck cancer before radiotherapy. The parameters of background and radiation-induced DNA damage, rate of repair, and residual non-repaired damage were measured by comet assay, and the repair kinetics for every donor were computer-fitted to an exponential curve. The level of background DNA damage before irradiation measured by comet assay as well as the level of micronuclei were significantly higher in the head and neck cancer patient group than in the healthy donors, while the parameters of repair were widely scattered in both groups. Cancer patient group contained significantly more individuals, whose irradiated lymphocytes showed high DNA damage, low repair rate and high non-repaired DNA damage level. Lymphocytes of donors belonging to this subgroup showed significantly lower inhibition of cell cycle after irradiation.     

Role of gene 59 of bacteriophage T4 in repair of UV-irradiated and alkylated DNA in vivo.

Nonsense mutants in gene 59 (amC5, amHL628) were used to study the role of this gene in the repair of UV-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to UV irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after UV irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to UV irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that UV repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B (su minus), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of UV lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules.     

The action of caffeine on X-irradiated HeLa cells. VIII. Recovery from potentially lethal damage

Recovery from potentially lethal radiation damage in HeLa S3 cells has been studied by irradiating synchronous cultures with 4 Gy at selected ages in the cell cycle, initiating treatment with 4 mM caffeine, which prevents recovery, at progressively later times up to 24-30 h after irradiation, and determining the plateau level of survival after incubation with the caffeine until 36-40 h after mitotic collection. Cell recovery appears to begin immediately after irradiation at any time during interphase: an accelerating increase in survival gives way after several hours to a linear increase which lasts for an additional several hours. The median recovery time is approximately 13 h after irradiation at any time during G1, but is markedly shorter (5-7 h) after irradiation in S or G2. The rate of recovery is slightly depressed if DNA replication is inhibited with aphidicolin after irradiation and slightly enhanced if protein synthesis is inhibited with cycloheximide. Both the rate and the extent of recovery are dependent on the location of the cells in the cycle at the time of irradiation--both functions increasing with cell age from the beginning of S, but having different age dependencies in G1. Blocking cell progression with a DNA-synthesis inhibitor before irradiation halts the age-dependent changes.     

Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5 degrees C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22 degrees C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.     

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P = 0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage.     

Spontaneous Mitotic Recombination and Evidence for an X-Ray-Inducible System for the Repair of DNA Damage in DROSOPHILA MELANOGASTER

Spontaneous mitotic recombination in the left arm of chromosome 3 was examined in both unirradiated control flies and sibs irradiated early in development by determining the sizes and frequencies of multiple-wing-hair (mwh) clones in the wing blade of heterozygous mwh/+ flies. Approximately 16% of the spontaneous mwh clones arise from events generating cells with normal division rates. The remaining 84% result from events generating cells with an average cell division rate one-third that of the surrounding cells; these are thought to result from events that generate aneuploid cells. Such clones probably arise from a failure correctly to repair spontaneous DNA damage. The frequency of spontaneous events late in development decreases significantly after irradiation as much as 150 hours earlier in development. The suppression of spontaneous events decreases with a longer period of time between irradiation and the final cell divisions in the wing blade. These results suggest the existence of a repair system for DNA damage in Drosophila that is induced by irradiation. The decrease in effect with time following irradiation could result from slow degradation or dilution by subsequent cell growth and division.     

The roles of DNA polymerases alpha, beta, and gamma in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents

The involvement of DNA polymerases alpha, beta, and gamma in DNA repair synthesis was investigated in subcellular preparations of cultured hamster and human cells. A variety of DNA damaging agents, including bleomycin, neocarzinostatin, UV irradiation, and alkylating agents, were utilized to induce DNA repair. The sensitivity of repair synthesis, as well as replicative synthesis and purified DNA polymerase beta activity, to inhibition by the DNA polymerase inhibitors dideoxythymidine triphosphate, aphidicolin, cytosine arabinoside triphosphate, and N-ethylmaleimide was determined. No evidence was obtained for a major role of polymerase gamma in any type of repair synthesis. In both hamster and human cells, the sensitivity of bleomycin- and neocarzinostatin-induced repair synthesis to ddTTP inhibition was essentially identical with that observed for purified polymerase beta, indicating these repair processes proceeded through a mechanism utilizing polymerase beta. Repair synthesis induced by UV irradiation and alkylating agents was not sensitive to ddTTP, indicating repair of these lesions occurred through a pathway primarily utilizing a different DNA polymerase; presumably polymerase alpha. However, replicative synthesis was much more sensitive to polymerase alpha inhibitors than was repair synthesis induced by UV irradiation or alkylating agents. Neither the amount of DNA damage nor the amount of induced repair synthesis influenced the degree to which the different DNA polymerases were involved in repair synthesis. The possibility that "patch size" or the actual type of DNA damage determines the extent to which different polymerases participate in DNA repair synthesis is discussed.     

Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase

Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.     

Use of the comet-FISH assay to demonstrate repair of the TP53 gene region in two human bladder carcinoma cell lines

The alkaline single-cell gel electrophoresis (comet) assay can be combined with fluorescence in situ hybridization (FISH) methodology to investigate the localization of specific gene domains within an individual cell. The position of the fluorescent hybridization spots in the comet head or tail indicates whether the sequence of interest lies within or in the vicinity of a damaged region of DNA. In this study, we used the comet-FISH assay to examine initial DNA damage and subsequent repair in the TP53 gene region of RT4 and RT112 bladder carcinoma cells after 5 Gy gamma irradiation. In addition to standard comet parameter measurements, the number and location of TP53 hybridization spots within each comet was recorded at each repair time. The results indicate that the rate of repair of the TP53 gene region was fastest during the first 15 min after damage in both cell lines. When compared to overall genomic repair, the repair of the TP53 gene region was observed to be significantly faster during the first 15 min and thereafter followed a rate similar to that for the overall genome. The data indicate that the TP53 domain in RT4 and RT112 cells is repaired rapidly after gamma irradiation. Furthermore, this repair may be preferential compared to the repair of overall genomic DNA, which gives a measure of the average DNA repair response of the whole genome. We suggest that the comet-FISH assay has considerable potential in the study of gene-specific repair after DNA damage.     

Role of p21WAF1 in the cellular response to UV

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.     

Role of p21WAF1 in the Cellular Response to UV

UV or gamma irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin-dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21 function.