Repair replication in Chinese hamster cells after damage from ultraviolet light

Abstract— After irradiation with u.v. light, Chinese hamster cells perform repair replication of their DNA. Small numbers of nucleosides are inserted into DNA, such that when BrUdR is used there is no detectable change in density. Repair replication begins immediately after irradiation, but it decelerates steadily and at least half is complete within 4 hr. Repair replication saturates above 200 ergs/mm² at a level which represents 0.055 per cent replacement of all thymine sites in 4 hr. Repair replication in mammalian cells, in contrast to that in microorganisms, does not appear to replace pyrimidine dimers excised from DNA in acid soluble form, and neither repair nor semiconservative replication discriminates between BrUdR and TdR.     

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.     

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.     

MULTIPLE EFFECTS OF FLUORESCENT LIGHT ON REPAIR OF ULTRAVIOLET-INDUCED DNA LESIONS IN CULTURED GOLDFISH CELLS

Abstract— It is known that fluorescent light illumination prior to UV irradiation (FL preillumination) of cultured fish cells increases photorepair (PR) ability. In the present study, it was found that FL preillumination also enhanced UV resistance of logarithmically growing cells in the dark. This enhancement of UV resistance differs from induction of PR because it was not suppressed by cycloheximide (CH) and it occurred immediately after FL preillumination. The effects of FL preillumination on repair of UV-induced DNA lesions in the dark were examined by an endonuclease-sensitive site assay to measure the repair of cyclobutyl pyrimidine dimers, and by enzyme-linked immunosorbent assay to quantitate the repair of (6-4) photoproducts. It was found that excision repair ability for (6-4) photoproducts in the genome overall was increased by FL preillumination. Moreover, a decrease in (6-4) photoproducts by FL illumination immediately after UV irradiation of the cells was found, the decrement being enhanced by FL preillumination with or without CH.     

DNA synthesis in cultured human fibroblasts: regulation by 3':5'-cyclic AMP.

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [3H] thymidine incorporation into acid-precipitable material, beginning after 8-12 hr and reaching maximum levels of 16-24 hr. Addition of dibutyryl-3':5'-cyclic AMP (DBcAMP) together with serum inhibited [3H] thymidine incorporation by 75-95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3':5'-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [3H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G1 was inhibited by high concentrations of 3':5'-cyclic AMP.     

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.     

INDUCIBLE POSTREPLICATION REPAIR IS RESPONSIBLE FOR MINIMAL MEDIUM RECOVERY IN UV-IRRADIATED Escherichia coliK–12

Abstract— Ultraviolet (UV)-irradiated Escherichia coli K–12 uvrA cells showed higher survival if plated on minimal growth medium rather than on rich growth medium, i.e., they showed minimal medium recovery (MMR). A 2-hour treatment of UV-irradiated cells with rifampicin inhibited the subsequent expression of MMR, and produced a large reduction in survival. We have recently isolated a new mutant ( mmrA1 ) that does not show MMR. The mmrA mutation protected UV-irradiated uvrA cells from the effect of rich growth medium on survival, but not from the effect of rifampicin on survival. DNA daughter-strand gap (DSG) repair in UV-irradiated (4 J/m²) uvrA cells was inhibited to the same degree whether rich growth medium was added immediately after irradiation or after 10 min of postirradiation incubation in minimal growth medium. However, chloramphenicol added immediately after irradiation greatly reduced this repair; there was less reduction if it was added 10 min after UV irradiation. These findings suggest that MMR is an inducible repair phenomenon, and that rich growth medium inhibits this repair process itself rather than its induction.     

ULTRAVIOLET-INDUCED CELL DEATH IS INDEPENDENT OF DNA REPLICATION IN RAT KANGAROO CELLS

Rat kangaroo (Potorous tridactylus) cells have an efficient repair system for photoreactivation of lethal lesions induced by 254 nm UV. However, this ability is lost with increasing time after UV, being completely ineffective after 24 h. Critical events leading to UV-induced cell death must occur within this period of time. DNA synthesis was inhibited by the DNA polymerase inhibitor aphidicolin and the loss of the capability to photorepair lethal lesions was maintained as for replicating cells. Similar data were obtained in synchronized cells UV irradiated immediately before S phase. Under the same conditions, the ability to remove cyclobutane pyrimidine dimers by photoreactivation in these cells remained unchanged 24 h after irradiation. These data indicate that the critical events responsible for UV-induced cell death occur in the absence of DNA replication.     

PYRIMIDINE DIMER SITES ASSOCIATED WITH THE DAUGHTER DNA STRANDS IN UV-IRRADIATED HUMAN FIBROBLASTS

Abstract. Pyrimidine dimer sites associated with the newly-synthesized DNA were detected during post-replication repair of DNA in UV-irradiated human fibroblasts. These pyrimidine dimer sites were inferred from a decrease in the molecular weight of pulse-labelled DNA after treatment with an extract of Micrococcus luteus containing UV-specific endonuclease activity. In DNA synthesized immediately after irradiation the frequency of these daughter strand dimer sites was 7–20% of that in the parental DNA. Such sites were found in fibroblasts from normal donors and from xeroderma pigmentosum patients (with defects in excision-repair or post-replication repair). They were excised from the DNA of normal cells. As the time between UV-irradiation and pulse-labelling was increased, the frequency of dimer sites associated with the labelled DNA decreased. If the pulse-label was delivered 6 h after irradiation of normal cells or excision-defective xeroderma pigmentosum cells, no dimer sites were detected in the labelled DNA. It has usually been assumed that daughter-strand dimer sites were the result of recombinational exchanges. The assay procedure used in these experiments and in similar experiments of others did not distinguish between labelled DNA containing pyrimidine dimers within the labelled section, and labelled DNA which did not contain pyrimidine dimers but was attached to unlabelled DNA which did contain dimers. The latter structures would arise during normal replication immediately following UV-irradiation of mammalian cells. Calculations are presented which suggest that a significant proportion and conceivably all of the dimer sites associated with the daughter strands may have arisen in this way, rather than from recombinational exchanges as has been generally assumed.     

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

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     

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.     

POSTIRRADIATION PROPERTIES OF A UV-SENSITIVE VARIANT OF CHO

Abstract A UV-hypersensitive mutant of Chinese hamster ovary (CHO) cells, termed 43-3B, has been used in a comparative study with the wild type CHO in order to determine the involvement of repair in several postirradiation phenomena. 43-3B has the same growth rate and chromosome number as the wild type CHO-9. It is hypersensitive to UV irradiation (D₀ of 0.3 J/m² as compared to 3.2 J/m² for the wild type). 43-3B shows only about 17% of the UV-stimulated unscheduled DNA repair synthesis of CHO-9 as measured by autoradiography. When breaks in supercoiled chromatin are measured after UV by the nucleoid sedimentation method, the mutant appears to be capable of carrying out only limited incision. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line, suggesting that the removal of UV-induced replication blocks by excision repair is the most important factor in allowing recovery of UV-inhibited DNA synthesis. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild type CHO-9, but little recovery was seen in 43-3B. This indicates that excision repair capability can also be important in split-fluence recovery.     

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.     

SKH-1 hairless mice repair UV-induced pyrimidine dimers in epidermal DNA

The SKH-1 hairless mouse strain has been used extensively as a model for human photocarcinogenesis, photoimmunology and photoaging, but little is known about DNA repair in living mouse skin. Mice were irradiated with UV-B light at doses which produce mild to severe sunburn, and the frequency of pyrimidine dimers in epidermal DNA was measured immediately and 6 h after irradiation using T4 endonuclease V treatment and alkaline agarose gel electrophoresis. The results demonstrate significant removal of pyrimidine dimers in mouse skin in vivo, with a dimer half-life of 7.4 h. These findings are similar to the repair of dimers in human skin in vivo. The SKH-1 hairless mouse is thus a useful model for pyrimidine dimer repair in human skin.     

Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.     

RANDOM DISTRIBUTION OF HIGHLY REPETITIVE AND INTERMEDIATE FREQUENCY MOUSE L-929 CELL DNA SEQUENCES SYNTHESIZED AFTER UV LIGHT EXPOSURE

Abstract— The DNA precursor ³H-thymidine incorporation rate (dpm/pg DNA) in mouse L-929 cells decreases immediately after exposure to UV light. This decrease is initially dose dependent, but at exposures greater than 22.5 J/m² appears to be radio-resistant. This was not explained by measurements of uptake of ³H-thymidine into the acid soluble pool (dpm/pg DNA). The radio-resistant incorporation amounted to approximately 35% of the control rate. DNA reassociation ("C₀T") studies were performed with DNA labeled with ³H-thymidine immediately after exposure of L-929 cells with a dose of UV resulting in radio-resistant incorporation to determine whether this radio-resistant incorporation was occurring in sequences of a particular repetitious frequency. These studies, performed in the highly repetitive to intermediate range, showed that the radio-resistant incorporation was occurring in DNA of all classes of repetitious frequency. DNA synthesized at different times after UV exposure, during the period when post-replication repair can occur, was similarly labeled for short intervals and isolated. The DNA reassociation studies showed that this DNA synthesis was also of all classes of repetitious frequency.     

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV‐light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis‐syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6‐4)pyrimidone photoproducts ((6‐4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6‐4)C lesion containing DNA duplex in complex with the (6‐4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6‐4) lesions into strained four‐membered ring repair intermediates, it is surprising that the not rearranged T(6‐4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6‐4) DNA photolyases.     

EFFECTS OF 8-METHOXYPSORALEN-INDUCED PHOTOTOXIC EFFECTS ON MAMMALIAN EPIDERMAL MACROMOLECULE SYNTHESIS IN VIVO

Abstract— The influence of 8-methoxypsoralen (8-MOP) and ultraviolet (UVA; 315–400 nm) radiation-induced phototoxic responses on DNA, RNA and protein synthesis and the DNA repair phenomenon were investigated utilizing the hairless mouse epidermis in vivo . The radioactive tracers TdR-³H, cytidine-³H and histidine-³H were used to examine changes in these macromolecules. Using these techniques, we found that the 8-MOP-UVA phototoxic injury inhibited premitotic semiconservative DNA synthesis in the germanitive layer of the epidermis within the first few hours post-irradiation. Recovery occurred by 24 h, followed by a progressive acceleration of this function over the next 7 days. No depression in RNA or protein formation was noted through 36 h. By 48 h the cells in the upper 1/2 to 1/3 of the epidermis lost their normal appearance and discontinued synthesizing these macromolecules. At 72 h RNA and protein synthesis was again active throughout the epidermis and the apparently dead cells had desquamated. At this time the epidermis was notably acanthotic and the epidermal cells were markedly enlarged. Examination for the dark repair response revealed no evidence of unscheduled DNA synthesis following irradiation indicating that the excision repair process was not demonstrable within the first 15min after the phototoxic injury. These responses differ in a number of parameters from the phototoxic reactions induced by UV rays shorter than 320 nm.