Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G₀ state. Autoradiography studies of cells released from G₀ after 72 h and replated at lower densities (3?9 × 10³ cells/cm²) in fresh medium containing 15% fetal bovine serum showed that semiconservative DNA synthesis (S phase) began ～24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G₀, allowed them to reattach at lower densities, irradiated them in early G₁ (～18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G₀, plated at cloning densities and irradiated as soon as they had attached and flattened out (～20 h prior to S) or 4, 8, 12, 16, 20 or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.     

Zinc-induced metallothionein synthesis by Caco-2 cells

Caco-2 cells possess many morphological and biochemical characteristics of intestinal absorptive cells, including the ability to transport zinc. In the present study, metallothionein (MT) synthesis in response to increased levels of zinc was examined. Increased incorporation of [³⁵S]cysteine into MTs was observed when excess ZnCl₂ was added to the medium. The rate of MT synthesis was found to be concentration dependent. Also, induction of MT synthesis was greater early in the culture, before the cells were fully differentiated. Incubation of the monolayers with ⁶⁵Zn and 200 μm zinc revealed that approximately 50% of the zinc incorporated into the cells was associated with MTs. The remainder was associated with large proteins as well as amino acids and small peptides. Actinomycin D and cycloheximide both inhibited the induction of MT synthesis, suggesting that the newly synthesized MTs are a result of expression of MT genes. Hence, Caco-2 cells, a model of intestinal absorptive cells, may be used to examine the role of MTs in zinc absorption.     

Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus

Summary The ability to reactivate ultraviolet (UV) damaged phage CbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage CbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2–2 h at 30°C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5–10 J/m² of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec-requiring DNA repair system analogous to W-reactivation in Escherichia coli. In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.     

Dose-dependent increase in repair of 1-β-d-arabinofuranosylcytosinedetectable DNA lesions in UV-treated xeroderma pigmentosum (group A) fibroblasts

The extent of DNA-excision repair was determined in human fibroblast strains from clinically normal and xeroderma pigmentosum complementation group A (XP-A) donors after irradiation with 254-nm ultraviolet (UV) light. Repair was monitored by the use of 1-β-d-arabinofuranosylcytosine (araC), a potent inhibitor of DNA synthesis, and alkaline sucrose velocity sedimentation to quantitate DNA single-strand breaks. In this approach, the number of araC-accumulated breaks in post-UV incubated cultures becomes a measure of the efficiency of a particular strain to perform long-patch excision repair. The maximal rate of removal of araC-detectable DNA lesions equalled ∼ 1.8 sites/10⁸ dalton/h in the normal strains (GM38, GM43), while it was more than 10-fold lower in both XP-A strains (XP4LO, XP12BE) examined. In normal fibroblasts the number of lesions removed during the first 4 h after irradiation saturated at ∼ 10 J/m². In contrast, the residual amount of repair in the excision-deficient cells increased as a linear function of UV fluence over a range 5–120 J/m². Thus we conclude that the repair of araC-detectable UV photoproducts in XP group A fibroblasts is limited by availability of damaged regions in the genome to repair complexes.     

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

The xeroderma pigmentosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F, were compared with normal human primary fibroblasts with regard to repair of damage induced by 254-nm UV. In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis = UDS), was about 50% of the value found in normal human cells. In these cells also the removal of UV-induced sites recognized by a specific UV-endonuclease proceeds at a reduced rate. By having BUdR incorporated into the repaired regions, followed by the induction of breaks in these patches by 313-nm UV, it was shown that the reduced repair synthesis is not caused by a shorter length of the repair regions in XP2RO, but is solely due to a reduction in the number of sites removed by excision repair. In XP23OS a discrepancy was observed between the level of UDS, which was about 10% of the normal value, and other repair-dependent properties such as UV survival, host-cell reactivation and removal of UV-endonuclease-susceptible sites, which were less reduced than could be expected from the UDS level. However, when UDS was followed over a longer period than the 2 or 3 h normally used in UDS analysis, it appeared that in XP23OS cells, the rate of UDS remained constant whereas the rate decreased in normal control cells. Consequently, the residual level of UDS varies with the period over which it is studied.     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

Transfer of copper from metallothionein to nonmetallothionein proteins in cultured cells

Copper uptake and distribution with time among cytoplasmic proteins were followed in cultured cells under several conditions: (1) CHO cells, which cannot synthesize metallothioneins, were labeled with⁶⁷Cu in the presence of 100 μM ZnCl₂; (2) Cd^r30F9 cells, which contain some constitutive metallothionein (MT), were labeled in the absence of additional ZnCl₂ and; (3) Cd^r30F9 cells were labeled in the presence of ZnCl₂, under which conditions they synthesized additional metallothioneins. The exogenous⁶⁷Cu and ZnCl₂, where present, were then removed, and the distributions of⁶⁷Cu among size fractions of the cellular proteins were observed at intervals for 16 h. In addition, a culture identical to condition (3) above was also treated with 100 μM ZnCl₂ during the redistribution period. The⁶⁷Cu was initially resolved into three peaks by Sephadex G-75 chromatography: high molecular weight, intermediate molecular weight, and MT. The⁶⁷Cu in the MT fraction decreased with at _1/2 of 10–12 h. In contrast to this, generally, in cells with a higher initial⁶⁷Cu bound to metallothionein, there was a progressive increase in the amount of⁶⁷Cu eluting with the high- and intermediate-molecular-weight fractions. Since no other source of⁶⁷Cu was available, these experiments suggest that copper stored in MT can be transferred to other proteins in these cells.     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

Regulation of the yeast RAD2 gene DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival

Summary In the yeast Saccharomyces cerevisiae the RAD2 gene is absolutely required for damage-specific incision of DNA during nucleotide excision repair and is inducible by DNA-damaging agents. In the present study we correlated sensitivity to killing by DNA-damaging agents with the deletion of previously defined specific promoter elements. Deletion of the element DRE2 increased the UV sensitivity of cells in both the G₁/early S and S/G₂ phases of the cell cycle as well as in stationary phase. On the other hand, increased UV sensitivity associated with deletion of the sequence-related element DRE1 was restricted to cells irradiated in G₁/S. Specific binding of protein(s) to the promoter elements DRE1 and DRE2 was observed under non-inducing conditions using gel retardation assays. Exposure of cells to DNA-damaging agents resulted in increased protein binding that was dependent on de novo protein synthesis.     

Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: Caffeine sensitive and caffeine resistant mechanisms

Replicative bypass repair of UV damage to DNA was studied in wide variety of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP)), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthetized after irradiation with 10 J/m² to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionall, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimidine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability.     

A comparison of the DNA binding,cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G_o state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.     

Repair and plasmid R46 mediated mutation requires inducible functions in Proteus mirabilis

Summary In Proteus mirabilis nalidixic acid or a predose of UV induce Rec protein formation, a portion of post-UV replication repair and post-UV replication enhancement. These inducible functions are not significantly affected by the plasmid R46, which renders P. mirabilis efficiently UV-mutable. The R46-mediated UV induction of rif ^r mutations requires additional inducible functions, as existing after malidixic acid treatment in rec ⁺ strains. After a nalidixic acid pretreatment UV efficient induction of rif ^r mutations occurs without an otherwise obligatory period of post-UV incubation prior to plating on rifampicin agar. The inducible character of this qualification of plasmid R46-mediated UV mutagenesis in P. mirabilis is evident from the inhibitory effects of chloramphenicol and starvation. Constitutive high-level synthesis of Rec protein in cells harboring the recombinant (multi-copy) rec ⁺ plasmid pPM1 reduced plasmid R46-mediated UV mutagenesis, probably by preventing (inducible?) functions required by the plasmid R46 repair-mutator.     

Effect of the adaptive response on the induction of the SOS pathway in E. coli K-12

Summary The adaptive-response is an inducible repair system of E. coli which reduces the mutagenic and cytotoxic effects of alkylation damage (Samson and Cairns, 1977). In adapted cells (cells exposed to sublethal doses of alkylating agents) the induction of W-reactivation and W-mutagenesis by alkylating agents is almost totally blocked. Despite the fact that adaptation has no detectable effect on UV mutagenesis in E. coli K-12, it does inhibit to some extent the UV and tif-1 mediated induction of SOS functions such as W-reactivation and prophage induction. Furthermore, the kinetics of induction of W-mutagenesis following UV treatment are altered by adaptation. In this case the adaptive-response seems to specifically block the induction of an error-producing W-reactivating capacity which normally would increase soon after UV treatment, while affecting error-free W-reactivating systems to a lesser extent.     

DNA excision in repair proficient and deficient human cells treated with a combination of ultraviolet radiation and acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide

Excision repair was measured in normal human and xeroderma pigmentosum group C fibroblasts treated with ultraviolet radiation and the carcinogens acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide (4NQO) by the techniques of unscheduled synthesis, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and assays of sites sensitive to ultraviolet (UV)-endonuclease. Doses of ICR-170 and 4NQO, low enough not to inhibit unscheduled DNA synthesis (UDS), caused damage to DNA that was repaired by a long patch type mechanism and the rates of UDS decreased rapidly in the first 12 h after treatment. Repair after a combined action of UV plus ICR-170 or UV plus 4NQO was additive in normal cells and no inhibition of loss of endonuclease sensitive sites was detected. In xeroderma pigmentosum (XP) C cells there was less repair after UV plus ICR-170 than after each treatment separately; whereas there was an additive effect after UV plus 4NQO and no inhibition of loss of endonuclease sensitive sites. The results indicate that in normal human fibroblasts there are different rate limiting steps for removal of chemical and physical damages from DNA and that XP cells have a different repair system for ICR-170, not just a lower level, than normal cells. Possibly the same long patch repair system works on 4NQO damage in both normal and XP cells.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

Comparative studies of host-cell reactivation, colony forming ability and excision repair after UV irradiation of xeroderma pigmentosum, normal human and some other mammalian cells

Host-cell reactivation, that is, the degree of survival of Herpes simplex virus after UV irradiation, was high in African green monkey BSC-1 cells, intermediate in normal human fibroblasts and human FL cells, and low in both xeroderma pigmentosum (XP) cells and mouse L cells. However, colony-forming ability after UV was high for FL, normal human fibroblasts and L cells, slightly low for BSC-1 cells and extremely low for XP cells. During the 24-h post-UV incubation period, up to about 50% of the thymine-containing dimers in the acid-insoluble DNA fraction disappeared at an almost equal rate for BSC-1, FL and normal human cells but remained unaltered for the XP cells. Alkaline sucrose gradient centrifugation of DNA after UV irradiation revealed only a slight difference between FL and BSC-1 cells in the kinetics of formation of single-strand breaks and their apparent repair. From these and the previously known characters of L cells possessing reduced excision-repair ability, if any, we may conclude that, if the survival of UV-irradiated Herpes simplex virus on a test line of human or other mammalian cells is as low as that on excisionless XP cells, then it is very probable that the test cell line is defective in excision repair. This reasoning leads to the presumptive conclusion that mouse L cells have an enhanced post-replication repair other than excision repair to deal with UV damage responsible for inactivation of colony-forming ability.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

UV-inducible repair: influence on survival, dimer excision, DNA replication and breakdown in Escherichia coli B/r Her+ cells.

Summary Using a model of double-UV-irradiation with inducing¹ (non-lethal) and lethal fluences² we have studied involvement of UV-inducible functions in post-UV-irradiation restoration processes and survival of Escherichia coli B/r thy ^- thy ^- Hcr⁺. Cells irradiated with both inducing and lethal fluences differed from cells irradiated with lethal fluence in the following respects: They were more UV resistant; they did not die during postincubation with chloramphenicol³; they exhibited a significant reduction in dimer excision; they were able to resume DNA replication and produce normal-sized DNA molecules in the presence of chloramphenicol. Since induction was provoked in cell prestarved for amino acids it was not associated with damage to points active in replication. However, the inducible product was more important for repair of replicating than non-replicating cells. The data indicate that protein necessary for resumption of DNA synthesis after UV is not constitutive but inducible.Abbreviations ¹IF inducing fluence - ²IF lethal fluence - ³CAP chloramphenicol     

Relation between repair mechanisms and induced mitotic recombination after UV irradiation, in the yeast Schizosaccharomyces pombe. Effects of caffeine

Summary Two different pathways A and 1 are known to control the repair of UV lesions in the yeast Schizosaccharomyces pombe. The relation between the UV-induced intergenic mitotic crossing over (MCO) and the repair of prelethal lesions controlled by these pathways were studied in the following strains: UVS1,₁/UVS1,₁, where pathway A acts; UVSA/UVSA where pathway 1 acts, UVS⁺/UVS⁺ (wild type) and UVS1A/UVS1A (double mutant). The analysis of the survival and MCO induction curves, and the comparison, as a function of the dose and as a function of survival, of the MCO induction curves corresponding to the different strains, show that the repair pathway 1 controls a mechanism involving recombination, and that the repair pathway A controls a mechanism which removes prerecombinational lesions. Studies were done with UVS1,₁/UVS1,₁ cells in different physiological conditions affecting the repair efficiency of prelethal lesions (irradiation during the logarithmic growth phase, liquid holding). In all cases the more efficient the repair of prelethal lesions is, the smaller is the recombination inducibility. This is expected if pathway A controls an excision repair mechanism.The effect of the repair inhibitor, caffeine, was studied. It inhibits only the repair of UV prelethal lesions controlled by pathway 1. The involvement of recombination in the repair of UV lesions in UVS⁺/UVS⁺ and UVSA/UVSA cells is also shown by the fact that the sensitization to the lethal effect of UV by caffeine in these strains is correlated with a decrease in UV MCO inducibility. Caffeine has no effect either on the UV survival, or on the MCO inducibility in UVS1,₁/UVS1,₁ cells. It is concluded that it inhibits the recombinational repair pathway and not the excision repair pathway.The MCO induction observed in UVS1/UVS1 and UVS1A/UVS1A cells could be due to the presence of a second recombinational pathway, not sensitive to caffeine. At least a fraction of the prerecombinational lesions would not be prelethal, and they are repairable by the excision repair mechanism.