Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

Genetic recombination of herpes simplex virus, the role of the host cell and UV-irradiation of the virus

Summary Recombination frequencies for two sets of genetic markers of herpes simplex virus were determined in various host cells with and without ultraviolet irradiation of the virus. UV irradiation increased the recombination frequency in all the cell types studied in direct proportion to the unrepaired lethal damage. In human skin fibroblasts derived from a patient with xeroderma pigmentosum (XP) of complementation group A, a given dose of UV stimulated recombination more than that in fibroblasts from normal individuals. On the other hand, UV stimulation of HSV recombination was slightly less than normal in fibroblasts derived from a patient with a variant form XP and from an ataxia telangiectasia patient. Caffeine, an agent known to inhibit repair of UV damage, reduced recombination in most of the cell types studied but did not suppress the UV-induced increase in recombination. These findings suggest that for virus DNA with the same number of unrepaired UV-lesions, each of the tested cell types promoted HSV-recombination to an equivalent extent.     

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.     

Host-cell reactivation of ultraviolet-irradiated SV40 DNA in five complementation groups of xeroderma pigmentosum.

Host-cell reactivation of UV-irradiated double-stranded SV40 DNA was studied in BSC-1 monkey cells, normal human cells, heterozygous Xeroderma pigmentosum (XP) cells, representative cell strains of the five complemention groups of XP and in XP "variant" cells. The following percentages of survival of the plaque-forming ability of double-stranded SV40 DNA were found in XP cells compared with the value found in normal monkey and human cells: group A, 13%; group B, 30%; group C, 18%; group D, 14%; group E, 59%; and in the heterozygous XP cells almost 100%. The survival in XP "variant" cells was 66%. The survival of single-stranded SV40 DNA in BSC-1 cells was much lower than that of double-stranded SV40 DNA in XP cells of complementation group A, which possibly indicates that some repair of UV damage occurs even in XP cells of group A.     

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Excision repair of UV- or benzo[a]pyrene diol epoxide-induced lesions in xeroderma pigmentosum variant cells is 'error free'

It is known that cells from one class of xeroderma pigmentosum (XP) patients, called XP variants, carry out excision repair of UV-induced DNA damage at a normal rate and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation, but are much more sensitive to the mutagenic effect of UV. To see if this hypermutability were the result of an 'error-prone', excision repair process, we irradiated fibroblasts derived from an XP variant patient, XP4BE, under conditions that allowed the cells various lengths of time for excision repair before the onset of DNA synthesis (S phase) and assayed the frequency of 6-thioguanine (TG)-resistant mutants. Cells synchronized by release from confluence (G0 state) and irradiated just prior to S phase showed a dose-dependent increase in mutants at very high frequencies; cells irradiated in early G1, approximately 12 h before the onset of S phase, showed frequencies 4 times lower. Cells irradiated in the G0 state and allowed 24 h or 48 h for excision repair before the onset of S phase showed still lower frequencies. A comparison of the relative rates of decrease in mutant frequency with time for excision repair before the onset of S phase in XP variant cells and normal human fibroblasts after a dose of 4 or 6 J/m2 showed that these were equal. However, for every time point, the frequency of mutants induced per dose of UV was significantly higher in the XP variant population than in the normal, suggesting that the XP variant cells have an abnormally error-prone process of replicating DNA on a template containing unexcised lesions or normal cells are by-passing many of such lesions using an error-free process. A similar comparative study in synchronized populations of XP4BE cells and normal cells, using the anti 7,8-diol-9,10-epoxide of benzo[a]pyrene, showed that excision repair prior to the onset of S phase also decreased the frequency of mutants induced in XP variant cells by this agent. But for every dose and time point, the frequencies induced in XP4BE cells and normal cells were identical. Thus, the hypermutability of the XP4BE cells was specific to UV radiation-induced DNA lesions.     

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.     

Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells

We investigated the lethal, UV killing-potentiating and repair-inhibiting effects of trivalent arsenic trioxide (As2O3) and pentavalent sodium arsenate (Na2HAsO4) in normal human and xeroderma pigmentosum (XP) fibroblasts. The presence of As2O3 for 24 h after UV irradiation inhibited the thymine dimer excision from the DNA of normal and XP variant cells and thus the subsequent unscheduled DNA synthesis (UDS): excision inhibitions were partial, 30-40%, at a physiological dose of 1 microgram/ml and 100% at a supralethal dose of 5 micrograms/ml. Correspondingly, As2O3 also potentiated the lethal effect of UV on excision-proficient normal and XP variant cells in a concentration-dependent manner, but not on excision-defective XP group A cells. Na2HAsO4 (As5+) was approximately an order of magnitude less effective in preventing all the above repair events than As2O3 (As3+) which is highly affinic to SH-containing proteins. The above results provide the first evidence that arsenic inhibits the excision of pyrimidine dimers. Partially repair-suppressing small doses of As2O3 (0.5 microgram/ml) and Na2HAsO4 (5 micrograms/ml) enhanced co-mutagenically the UV induction of 6-thioguanine-resistant mutations of V79 Chinese hamster cells. Thus, such a repair inhibition may be one of the basic mechanisms for the co-mutagenicity and presumably co-carcinogenicity of arsenic. XP group A and variant strains showed a unique higher sensitivity to As2O3 and Na2HAsO4 killing by a yet unidentified mechanism.     

Effects of DNA damaging agents on cultured fibroblasts derived from patients with Cockayne syndrome.

The cytotoxic action of physical and chemical agents on 10 skin fibroblast strains in culture derived from individuals with Cockayne's syndrome was measured in terms of colony-forming ability. As compared to fibroblasts from normal donors, all Cockayne cell strains tested exhibited a significantly increased sensitivity to UV light and a normal sensitivity to X-rays. Cells from two sets of parents of unrelated Cockayne children showed an intermediate level of UV sensitivity. There was no effect of 0.5 mM caffeine on UV survival in normal and two Cockayne strains tested, indicating that postreplicational repair in Cockayne cells as measured by caffeine sensitivity was probably normal. Sensitivity of normal and Cockayne cells to the chemical carcinogens and mutagens 4NQO, N-AcO-AAF, ICR-170 and EMS was also compared. An increased sensitivity of Cockayne cells to 4NQO or N-AcO-AAF, but not the ICR-170 or EMS, was observed. However, unlike the intermediate UV sensitivity, the cell strains from two parents of Cockayne patients showed the same sensitivity to N-AcO-AAF or 4NQO as fibroblasts from normal individuals. Quantiation of damage to the DNA after 20 J . m-2 UV irradiation indicates normal levels of [3H] thymidine incorporation in the Cockayne cells, in contrast to UV-irradiated xeroderma pigmentosum cells (XP 12BE) in which there was a very low level of repari synthesis. Moreover, we have shown previously that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.     

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.     

Relationship between pyrimidine dimers, 6-4 photoproducts, repair synthesis and cell survival: studies using cells from patients with trichothiodystrophy

Trichothiodystrophy is a genetic disease which in the majority of cases studied is associated with a deficiency in the ability to repair UV damage in cellular DNA. Three categories of UV response have been identified. In type 1 the response is completely normal, whereas type 2 cells are deficient in excision-repair, with properties indistinguishable from those of XP complementation group D. Type 3 cells have normal survival following UV-irradiation and normal rates of removal of cyclobutane pyrimidine dimer sites. Nevertheless repair synthesis is reduced by 50% in these cell strains and this is associated with a marked reduction in the repair of 6-4 photoproducts from cellular DNA. The present results show that 50% or more of repair synthesis at early times after irradiation of normal primary human fibroblasts is attributable to repair of 6-4 products. They also suggest that repair of cyclobutane dimers is crucial for cell survival.     

Differential repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in DNA of human fibroblasts exposed to ultraviolet light and 4-nitroquinoline 1-oxide

The extent of DNA excision repair was determined in dermal fibroblast strains from clinically normal and xeroderma pigmentosum (XP; complementation group A) human donors after single or combined exposures to 254-nm ultraviolet light and 4-nitroquinoline 1-oxide (4NQO). The repair was monitored by incubation of the treated cultures in the presence of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of long-patch excision repair, followed by quantitation of araC-accumulated DNA single-strand breaks (representing repair events) by velocity sedimentation analysis in alkaline sucrose gradients. The amount of repair in normal fibroblast strains increased as a function of UV fluence and reached a plateau at 15 J/m2; strand breaks were not detected when these same cultures were irradiated with as much as 60 J/m2 UV and incubated in the absence of araC, implying that an initial (incision) step is rate-limiting in the repair of UV damage. In normal fibroblasts (i) the incidence of araC-detectable lesions removed during fixed intervals following exposure to 4NQO (4 microM; 30 min) was approximately 2.5 times greater than that seen following irradiation with repair-saturating fluences (greater than or equal to 15 J/m2) of UV-rays; and (ii) the amount of repair in cultures treated simultaneously with 4NQO (0.5-6 microM; 30 min) and a repair-saturating fluence of UV (20 J/m2) was found to approach the sum of that arising from exposure to each separately. The XP cells (XP12BE) exhibited a deficiency in the removal of araC-detectable DNA lesions following exposure to either of the carcinogens. Since araC is known to inhibit the repair of alkali-stable 4NQO-DNA adducts (i.e., lesions assumed to be removed by the UV-like excision pathway) but not that of alkali-labile sites (i.e., DNA lesions operated on by the X-ray-like repair pathway), our results strongly imply that the multistep excision-repair pathway operative on UV photoproducts in human fibroblasts differs from that responsible for removing alkali-stable (araC-detectable) 4NQO adducts by at least one step, presumably the rate-limiting incision reaction mediated by a lesion-recognizing endonuclease.     

Infection of UV-irradiated xeroderma pigmentosum fibroblasts by herpes simplex virus: study of capacity and Weigle reactivation.

The capacity of monolayers of both normal human and xeroderma pigmentosum (XP) filbroblasts to support plaque formation by herpes simplex virus was decreased when the monolayers were ultraviolet (UV) irradiated and infected with virus. Fibroblasts of XP complementation groups A, B, and D were sensitive to UV, being 4-6 fold more sensitive than either fibroblasts of XP complementation group C or fibroblasts from a normal individual. When the monolayers were irradiated 4 days prior to infection, the capacity of normal fibroblasts to support herpes virus growth recovered, whereas the capacity of the XP strains decreased further compared to that measured when infection immediately followed irradiation. Concurrent experiments with UV-irradiated herpes virus showed that the survival of this virus did not increase when infection by irradiated virus immediately followed irradiation of the monolayers. However, if the monolayers were irradiated 4 days prior to infection, the survival of this virus increased by a factor of nearly 2. Such Weigle reactivation (WR) occurred at lower fluences to the XP fibroblasts than to normal fibroblasts, suggesting that WR results from residual cellular DNA damage left after excision repair.     

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.     

A comparison of the response of unstimulated and stimulated T-lymphocytes and fibroblasts from normal, xeroderma pigmentosum and trichothiodystrophy donors to the lethal action of UV-C.

Unstimulated T-lymphocytes from normal donors are significantly more sensitive to the lethal effects of UV-C than either stimulated T-lymphocytes or fibroblasts as judged by colony-forming ability. Data from other studies suggest that excision repair is more effective in stimulated than unstimulated T-lymphocytes leading to the prediction that these differences in survival should be minimal in cells established from excision defective donors. The prediction was met with XP6BR, a donor of unknown complementation group. For 3 XP's from complementation group D, however, enhanced survival in stimulated T-cells was observed. With cells from an excision-defective TTD who was included in complementation group D of XP both fibroblasts and unstimulated T-lymphocytes were hypersensitive. For a second excision defective TTD patient who was excluded from complementation group D, the unstimulated T-lymphocytes were more resistant than those of normal donors although the fibroblasts were hypersensitive. These results suggest that the in vitro response of stimulated T-lymphocytes or fibroblasts may not reflect the in vivo response of cells, as measured by the response of unstimulated T-lymphocytes.     

Kinetic analysis of UV-induced incision discriminates between fibroblasts from different xeroderma pigmentosum complementation groups, XPA heterozygotes and normal individuals

The capacity of a variety of human fibroblasts to incise DNA following exposure to far ultraviolet-light is determined from the rate of single-strand DNA break accumulation in the presence of DNA synthesis inhibitors. We have quantitated incision, one of the early steps in the UV excision repair pathway, in cells form normal, xeroderma pigmentosum groups C, D, G, H and variant individuals, and in the parents of one XPA patient. On the basis of the estimated initial rates of incision the different XP cells examined in this work can be ranked as follows: XP variant much greater than XPH greater than XPH greater than XPD greater than XPC greater than XPG greater than XPA. In each cell strain breaks accumulate immediately after irradiation over a range of 0.5-20 Jm-2 with the exception of the XPC strain examined, where there is an initial delay of 15 min. The rate of incision in XPA heterozygote cells is roughly half that of normal fibroblasts. Analysis of the kinetics of break accumulation over short intervals after irradiation permits estimation of the apparent enzymatic parameters, Km and Vmax, for the incision step. The approximate values of Km and Vmax for normal and XP variant are similar while for the heterozygotes of an XPA individual Km values are normal (around 1 Jm-2), but there is only half the amount of normal enzyme activity. XPD and H cells express low levels of active enzyme, between 5 and 15% of that of the normal, but while the Km of XPH is very similar to that of normal cells, that of two XPD strains examined is between 2- and 3-fold higher.     

Caffeine inhibition of the repair of ultraviolet-irradiated adenovirus in human cells.

Caffeine is shown to block repair of ultraviolet-irradiated adenovirus 2 when the irradiated virus infects normal human fibroblasts from a xeroderma pigmentosum (XP) variant. Such blockage is not observed when the irradiated virus infects XP fibroblasts belonging to XP complementation group A. Thus normal and XP variant cells have a caffeine-sensitive repair process. This may be either excision or an excision dependent repair process because fibroblasts belonging to XP complementation group A are believed to lack the excision repair process.     

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.     

Dose-dependent increase in repair of 1-beta-D-arabinofuranosylcytosine-detectable 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-beta-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 approximately 1.8 sites/10(8) 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 approximately 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.