THE TIME SEQUENCE OF EVENTS DURING EXCISION REPAIR IN TETRAHYMENA PYRIFORMIS

Abstract— The time sequence of events during excision repair of DNA in Tetrahymena pyriformis was investigated after sublethal dose of u.v. radiation. Buoyant-density analysis of the DNA from repairing cells grown in medium containing 5'-bromodeoxyuridine makes it possible to distinguish repair synthesis from normal synthesis. Analysis of the DNA synthesized at various times after irradiation clearly indicates that repair synthesis starts very quickly after irradiation and is completed within 3 to 4 hr. Immediately after irradiation, normal DNA synthesis is greatly depressed but gradually resumes until it predominates after 3 to 4 hr. The molecular weight of DNA strands is reduced and the net rate of DNA synthesis is depressed immediately after irradiation. Both of these parameters are restored to their pre-irradiation values by 3 to 4 hr after irradiation. During the repair period the majority of the pyrimidine dimers are removed. All of the data indicate that repair begins immediately after irradiation and is completed in 3 to 4 hr (about two thirds of a generation period).     

The effect of ultraviolet light on RNA metabolism in bromouracil deoxyriboside-grown HeLa cells

Abstract— The presence of 5-bromouracil deoxyriboside (BrUdR) in the DNA of HeLa cells has profound effects on RNA metabolism after u.v. irradiation. In normally grown cells 200 ergs/mm² depresses RNA synthesis by about 30 per cent while in BrUdR-grown cells the same exposure to u.v. depresses RNA synthesis by 95 per cent. When BrUdR-grown cells are u.v. -irradiated after being labeled with ³H-uridine, the normal autoradiographic pattern, where label shifts from nucleus to cytoplasm, fails to occur. Also, in lieu of the increase in RNA specific activity that is observed in unirradiated cells for a few hours after 20-min pulse-labeling, there occurs a constant decrease in specific activity after the irradiation.     

Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks

DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of gamma-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for > 4 h in addition to those induced at replication.     

EFFECTS OF DOSE FRACTIONATION ON ULTRAVIOLET SURVIVAL OF ESCHERICHIA COLI

Abstract— Exposure of E. coli B/r and B at low average dose rates of u.v. radiation (2537 Å), produced either by fractionated doses or by continuous irradiation at a very low dose rate (80 ergs/mm²/hr), results in much increased survival compared to single exposure at high dose rate. This increase is attributed to repair taking place during the irradiation period. The effect is small in the repair-deficient strains E. coli B_8-1_, and C syn- , and is absent in phage T1 and T4, which cannot undergo repair in the extracellular state. However, the prolonged time available for repair in these experiments accounts for only a very minor part of the increase in survival. The principal factor apparently is that the number of lesions present at any time remains relatively low. Presumably complete repair, not only the excision step, can occur in buffer during the irradiation period. This interpretation is supported by experiments in which cells were exposed to combinations of highly fractionated irradiation and single-dose irradiation. We therefore propose that mutual interference in repair, possibly by overlapping of repair regions in complementary DNA strands, reduces considerably the repair efficiency if many lesions are present. This hypothesis explains the 'shouldered' survival curves of B/r and possibly other E. coli strains as due to decreasing repair efficiency with increasing u.v. dose     

HOECHST 33258 DYE GENERATES DNA-PROTEIN CROSS-LINKS DURING ULTRAVIOLET LIGHT-INDUCED PHOTOLYSIS OF BROMODEOXYURIDINE IN REPLICATED AND REPAIRED DNA

Abstract— Substitution of bromodeoxyuridine for thymidine in the DNA of mammalian cells sensitizes them to a range of wavelengths of ultraviolet light. Cells are also sensitized to photochemical reactions involving dyes such as Hoechst 33258, which is used to produce differential staining of chromatids according to their bromodeoxyuridine content. Irradiation with 313 nm light of human and hamster cells containing bromodeoxyuridine in their DNA produced single-strand breaks, detectable by alkaline elution, but no DNA-protein cross-links. Irradiation with 360 nm light in the presence of Hoechst 33258 produced extensive DNA-protein cross-linkage as well as single-strand breaks. These cross-links were observed in DNA containing bromodeoxyuridine incorporated by either semiconservative or repair replication, and may provide a method for identification of proteins in close proximity to replication forks or repair sites. When the protein was removed with proteinase K, bromodeoxyuridine in repair patches after irradiation by doses of ultraviolet (254 nm) light as low as 0.26 J/m² could readily be detected. Hoechst 33258-mediated photolysis, therefore, provides a sensitive new technique for measuring repair replication after ultraviolet light irradiation.     

PHOTOREACTIVATION and EXCISION REPAIR OF UV INDUCED PYRIMIDINE DIMERS IN THE UNICELLULAR CYANOBACTERIUM GLOEOCAPSA ALPICOLA (SYNECHOCYSTIS PCC 6308)

Abstract— The survival curve obtained after UV irradiation of the unicellular cyanobacterium Synecho-cystis is typical of a DNA repair competent organism. Inhibition of DNA replication, by incubating cells in the dark, increased resistance to the lethal effects of UV at higher fluences. Exposure of irradiated cells to near ultraviolet light(350–500 nm) restored viability to pre-irradiation levels. In order to measure DNA repair activity, techniques have been developed for the chromatographic analysis of pyrimidine dimers in Synechocystis. The specificity of this method was established using a haploid strain of Sacchar-omyces cerevisiae. In accordance with the physiological responses of irradiated cells to photoreactivating light, pyrimidine dimers were not detected after photoreactivation treatment. Incubation of irradiated cells under non-photoreactivating growth conditions for 15 h resulted in complete removal of pyrimidine dimers. It is concluded that Synechocystis contains photoreactivation and excision repair systems for the removal of pyrimidine dimers.     

THE ACTION SPECTRUM AND DOSE RESPONSE STUDIES OF UNSCHEDULED DNA SYNTHESIS IN NORMAL HUMAN FIBROBLASTS

Abstract— Excision repair of DNA damage by UV has been assessed in normal human fibroblasts in culture by measuring unscheduled DNA synthesis. Dose response experiments indicated that the same chromophore was involved in UV-induced damage and excision repair at three different wavelengths between 260 and 300 nm. Action spectra for unscheduled DNA synthesis were determined at wavelengths between 260 and 320 nm 30 min after irradiation using 2 doses of UV, 100 J m^-2and 10Jm^-2. Experiments at the lower dose were carried out because it appeared that repair was saturated with the higher dose at 260 and 280 nm. To explore this part of the spectrum further, experiments were performed with different doses at 260 and 280 nm and unscheduled DNA synthesis assessed 30 min and 24 h after irradiation. At 24 hr after irradiation a significantly greater amount of unscheduled DNA synthesis occurred at 280 nm. It is suggested, therefore, that both DNA and protein are concerned in the absorption of UV which leads to DNA damage and excision repair.     

DETECTION OF UVR-INDUCED DNA DAMAGE IN MOUSE EPIDERMIS in vivo USING ALKALINE ELUTION

Abstract— Alkaline elution has been used to detect ultraviolet radiation (UVR)-induced DNA damage in the epidermis of C3H/Tif hr/hr mice. This technique detects DNA damage in the form of single-strand breaks and alkali-labile sites (SSB) formed directly by UVA (320–400 nm) or indirectly by UVB (280–320 nm). The latter induces DNA damage such as cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4)-photoproducts, which are then converted into transient SSB by cellular endonucleases, during nucleotide excision repair (NER). The irradiation system used had a spectral output similar in effect to solar UVR, with the UVB component inducing 94% of the edema response observed in mice. Consequently, the majority of SSB detected were those formed via NER of UVB-induced photoadducts. The number of SSB detected immediately after 8 kj/m² (2.7 minimum erythema doses determined at 48 h post-UVR [MED]) was low, indicating the formation of only small numbers of transient SSB. When DNA repair inhibitors hydroxyurea and 1 -β-D-arabinofuranosylcytosine were administered (intraperi-toneally) to mice 30 min before UVR, they prevented sealing of the DNA SSB formed during NER. A four-fold increase in the number of SSB detected resulted, which was found to be linearly related to the UVR dose. The SSB induced by 2 kj/m² (less than an MED) were readily detected, with the ear showing lower numbers of SSB than the dorsum. When repair inhibitors were added post-UVR, the rate of formation of SSB declined rapidly with time of administration, reflecting repair of DNA lesions. After a UVR dose of 6 kj/m² (2 MED), 50% of the initial repair-dependent SSB had been removed after approximately 2 h in the ear and 4 h in the dorsum; no more SSB appeared to be incised by 24 h post-UVR. The technique described is an efficient and highly sensitive one for the quantification of SSB induced in UV-irradiated skin samples in vivo.     

DNA REPAIR IN V-79 CELLS TREATED WITH COMBINATIONS OF PHYSICAL AND CHEMICAL CARCINOGENS*

Excision repair of DNA damage was measured by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair in Chinese hamster V-79 cells treated with 254 nm of ultraviolet radiation (UV), 7,12-dimethylbenz[a]anthracene 5,6-oxide (DMBA-epoxide), N-acetoxy-2-acetylaminofluorene (AAAF), 4-nitroquinoline 1-oxide (4NQO), 2-methoxy-6-chloro-9-[3(ethyl-2-chloroethyl)-aminopropylamino]acridine dihydrochloride (ICR-170), X-rays, ethylmethanesulfonate (EMS), methyl methanesulfonate (MMS) and combinations of these agents. Compared to normal human cells V-79 were defective in repair of UV lesions and the lesions induced by the UV-mimetic chemicals. The extent of the defects varied from 10 to 50% and was similar to those in Xeroderma pigmentosum group C cells (XP C). V-79 cells repaired X-ray damage and damage from the alkylating agents EMS and MMS to the same extent as human cells. Repair was additive after a combination of UV plus MMS indicating, as expected, that there are different rate-limiting steps for removal of the damages from these agents. Repair was less than additive in cells treated with UV plus ICR-170, AAAF plus ICR-170, AAAF plus 4NQO, and 4NQO plus ICR-170 and approximately equal to that observed for the higher of the two agents separately, indicating that there may be similar rate-limiting steps for removal of lesions. Although the results on repair after combinations of UV plus 4NQO, UV plus DMBA-epoxide or X-rays plus MMS were difficult to interpret, there was not any inhibition of repair in these combinations.     

PHOTODYNAMIC DAMAGE AND ITS REPAIR IN Vibrio cholerae

Abstract— Repair of photodynamic damage induced by acriflavine and visible light has been examined in three strains of Vibrio cholerae differing in their capabilities to repair ultraviolet (UV) light induced DN A damage. Excision repair deficient wild type cells of strain 154 are more sensitive to photodynamic treatment compared to repair proficient cells of strain 569B. However, no difference in their capabilities to repair of damage following photodynamic treatment can be detected. No single-strand breaks in the irradiated cell DNA are observed when the cell survival is more than 10%. Single-strand breaks observed at cell survival less than 5% are not dark repairable even in excision repair proficient wild type cells. Repair of membrane damage can partially account for the recovery observed at low doses. In contrast, radiation-sensitive mutant 569B_s cells which lack both excision and medium-dependent dark repair for UV-lesions are most efficient in repairing damage induced by photodynamic treatment.     

DNA REPAIR IN ULTRAVIOLET LIGHT IRRADIATED HeLa CELLS AND ITS REVERSIBLE INHIBITION BY HYDROXYUREA

Abstract— –The repair of u.v. damaged DNA in HeLa cells can be detected using the alkaline sucrose gradient technique. As a result of pyrimidine dimer excision single strand breaks are produced in DNA of irradiated cells. Rejoining of these breaks occurs during an 8 hr post-irradiation incubation period and is prevented by hydroxyurea and acriflavine. The inhibition of repair by hydroxyurea can be reversed by a mixture of all 4 deoxyribonucleosides at a concentration that does not reverse the inhibition of total DNA synthesis.     

PHOTOREACTIVATION OF ULTRAVIOLET IRRADIATED NON-DIVIDING POPULATIONS OF ICR 2A FROG CELLS

Abstract— Ultraviolet (UV) irradiation of non-dividing populations of ICR 2A frog cells led to their detachment from the surface of the culture dish and eventual lysis. Exposure of the cells to photoreactivating light after UV irradiation prevented cell killing and was accompanied by a loss of endonuclease sensitive sites from DNA. This photoreversal did not take place when the cells were exposed at 4°C to photoreactivating light indicating that the reversal was the result of photoenzymatic repair. As the action of photoreactivating enzyme is specific for the repair of pyrimidine dimers in DNA, these results suggest that pyrimidine dimers in DNA are the critical lesions leading to the death of non-dividing populations of UV irradiated cells.     

REPAIR OF PSORALEN PLUS NEAR ULTRAVIOLET LIGHT DAMAGE IN BACTERIOPHAGES T3 AND T4

Abstract— Repair of T3 and T4 DNA damaged by treatment with 8-methoxypsoralen plus near UV (PNUV) has been investigated. It is shown that the excision repair mechanisms of the host cell can repair a substantial fraction of the psoralen-DNA mono-adducts in T3 DNA, but cannot by themselves repair crosslinks. In contrast neither the excision repair system of the host nor the phage coded v gene endonuclease is involved in the repair of psoralen adducts in T4 DNA. Multiplicity reactivation is effective in the recovery of T4 DNA containing psoralen-DNA mono-adducts, but is ineffective in the recovery of crosslinked phages. Comparisons of the lethality of PNUV treatment and the number of crosslinks induced in T4 DNA show clearly that mono-adducts are lethal to this phage. Both T3 and T4, however, appear to effectively repair many mono-adducts by postreplicational repair.     

THE U.V. SENSITIVITY OF BACTERIA: ITS RELATION TO THE DNA REPLICATION CYCLE

Abstract— A striking increase in the shoulder of the u.v. survival curve but no change in the limiting slope is obtained when cultures of Escherichia coli strain TAU complete the DNA replication cycle in the absence of concommitant protein synthesis prior to irradiation. The u.v. sensitivity of protein synthesis or RNA synthesis is not altered significantly by this treatment.
In contrast to the result for strain TAU, there is no significant change in the u.v. survival curve for the u.v. sensitive E. coli B_s-1 when its DNA replication cycle is completed under similar conditions.
Following a period of inhibited protein synthesis there is a delay in the reinitiation of the normal DNA replication cycle when protein synthesis resumes. This delay would allow time for an intracellular repair system to operate before the attempted resumption of normal replication. Strain B_s-1, which is deficient in this repair system, would not be expected to benefit from such a delay, as consistent with the observed results. A model is presented to account for lethality due to attempted DNA replication during a period of repair synthesis. The maximum survival for a given u.v. dose would be predicted for a culture which has completed the normal DNA replication cycle prior to irradiation and which is not permitted to reinitiate the cycle until all possible repair synthesis is completed.     

Assessment of gamma ray-induced DNA damage in Lasioderma serricorne using the comet assay

We attempted a DNA comet assay under alkaline conditions to verify the irradiation treatment of pests. Lasioderma serricorne (Fabricius) were chosen as test insects and irradiated with gamma rays from a ⁶⁰Co source at 1 kGy. We conducted the comet assay immediately after irradiation and over time for 7 day. Severe DNA fragmentation in L. serricorne cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. The parameters of the comet image analysis were calculated, and the degree of DNA damage and repair were evaluated. Values for the Ratio (a percentage determined by fluorescence in the damaged area to overall luminance, including intact DNA and the damaged area of a comet image) of individual cells showed that no cells in the irradiated group were included in the Ratio<0.1 category, the lowest grade. This finding was observed consistently throughout the 7-day post-irradiation period. We suggest that the Ratio values of individual cells can be used as an index of irradiation history and conclude that the DNA comet assay under alkaline conditions, combined with comet image analysis, can be used to identify irradiation history.     

Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.     

DNA EXCISION REPAIR IN LYMPHOCYTES FOLLOWING PHOTOAFFINITY LABELING WITH ETHIDIUM MONOAZIDE

Irradiation of ethidium monoazide by fluorescent light promotes a chemical decomposition of the azide into a highly reactive nitrene intermediate. Covalent bonding of this electrophile to the DNA in the cell provokes repair of damage which can be monitored by incorporation of [³H]-thymidine. Human lymphocytes were labeled with [¹⁴C]-ethidium azide and then allowed to undergo DNA repair. Repair incorporation of [³H]-thymidine showed saturation at 5 µM ethidium azide, but excision of the labeled drug failed to saturate at 20 µM, suggesting that excision and resynthesis are two separate events. Cells were also labeled with the photosensitive drug and/or exposed to UV radiation, and then allowed to undergo a period of DNA repair. The tritium incorporation for the combined insults was less than the sum of the two insults. Quinacrine, progesterone and chloroquine inhibited repair incorporation of [³H]-thymidine, but had no effect on the excision of the drug from the DNA. After damage by ethidium azide, chromatin was isolated from lymphocytes which had been allowed to repair label with [³H]-thymidine. Partial digestion of the chromatin with micrococcal nuclease released 80% of the tritium when approximately 40% of the DNA had been hydrolyzed by the enzyme.     

THE BIOLOGY OF THE (6–4) PHOTOPRODUCT

The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.     

LIGHT ACTIVATION OF A COMPLEX MIXTURE: EFFECTS OF UV EXCISION REPAIR ON THE MODULATION OF GENOTOXIC AND MOLECULAR EVENTS IN CHINESE HAMSTER CELLS

Abstract— An aqueous effluent produced during the retorting of oil shale has been shown to induce a significant genotoxic response in cultured Chinese hamster (CHO) cells following activation by near ultraviolet light (UVA). In this report the light-activated responses induced by this complex mixture were compared between two DNA excision repair deficient mutants and their parental strain, CHO-AA8-4. The mutants, UV-5 and UV-135, were hypersensitive to both the cytotoxic and mutagenic effects of concurrent exposures to the retort process water and UVA. Repair proficiency appeared to render AA8-4 relatively insensitive to low doses of UVA treatment, whereas in the two mutants a linear dose-response in the induction of 6-thioguanine resistant (6TG^®) mutations was observed even at the lower UVA doses examined. Filter DNA alkaline elution methods were utilized to demonstrate that both single-strand breaks and DNA-DNA interstrand crosslinks were induced in CHO DNA following process water and UVA treatment. Results were also obtained which indicated that the inability to repair DNA-DNA crosslinks contributed significantly to the hypersensitive response seen in the excision repair mutants following the photoactivation of this complex mixture.     

Deficient Nucleotide Excision Repair in Squamous Cell Carcinoma Cells

Squamous cell carcinomas (SCCs) are associated with ultraviolet radiation and multiple genetic changes, but the mechanisms leading to genetic instability are unclear. SCC cell lines were compared to normal keratinocytes for sensitivity to ultraviolet radiation, DNA repair kinetics and DNA repair protein expression. Relative to normal keratinocytes, four SCC cell lines were all variably sensitive to ultraviolet radiation and, except for the SCC25 cell line, were deficient in global repair of cyclobutane pyrimidine dimers, although not 6‐4 photoproducts. Impaired DNA repair of cyclobutane pyrimidine dimers was associated with reduced mRNA expression from XPC but not DDB2 genes which each encode key DNA damage recognition proteins. However, levels of XPC or DDB2 proteins or both were variably reduced in repair‐deficient SCC cell lines. p53 levels did not correlate with DNA repair activity or with XPC and DDB2 levels, but p63 levels were deficient in cell lines with reduced global repair. Repair‐proficient SCC25 cells depleted of p63 lost XPC expression, early global DNA repair activity and UV resistance. These results demonstrate that some SCC cell lines are deficient in global nucleotide excision repair and support a role for p63 as a regulator of nucleotide excision repair in SCCs.