Repair of DNA damaged by mutagenic metabolites of benzo(a)pyrene in human cells

The repair of human DNA after damage by known and potential metabolites of benzo(a)pyrene has been examined utilizing the bromodeoxyuridine photolysis assay. Repair was characterized as either ultraviolet (“long”) or ionizing radiation type (“short”) repair utilizing normal cells and cells deficient in ultraviolet-type repair endonuclease from a patient with xeroderma pigmentosum (XP). We have found that only (±)-7β,8-dihydroxy-9β,-10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 1) and its disastereomer, (±)-7β,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 2) elicit damage to DNA which is recognizable by the ultraviolet excision repair system in normal human cells. Benzo(a)pyrene 4,5-, 9,10-, 11,12-oxides do not elicit damage which is repairable by this repair system. The 1,2-diol-3,4-epoxides from naphthalene have no measurable activity in our assay. These results indicate that both the benzo(a)pyrene ring structure and the diol epoxide groups are important in causing the damage to DNA which is repairable by the ultraviolet excision repair system. These results parallel the reported high mutagenic activity of these compounds and support the concept that benzo(a)pyrene 7,8-diol-9,10-epoxides may be the ultimate, metabolically activated forms of benzo(a)pyrene.     

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.     

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.     

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.     

Repair of UV-endonuclease-susceptible sites in the 7 complementation groups of xeroderma pigmentosum A through G

7 strains of human primary fibroblasts were chosen from the complementation groups A through G of xeroderma pigmentosum; these strains are UV-sensitive and deficient in excision repair of UV damage on the criterion of unscheduled DNA synthesis (UDS). They were compared with normal human fibroblasts and one xeroderma pigmentosum variant with regard to their capacity to remove pyrimidine dimers, induced in their DNA by UV at 253.7 nm. The XP variant showed a normal level of dimer removal, whereas 6 of the other XP strains had a greatly reduced capacity to remove this DNA damage, in agreement with their individual levels of UDS. Strain XP230S (complementation group F), however, only showed a 20% reduction in the removal of dimers, which is much less than expected from the low level of UDS in this strain.     

Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis

Repair of ultraviolet-irradiated transforming deoxyriboinucleic acid (DNA) in several strains of Bacillus subtilis was studied in order to determine the effects of excision repair and postreplication repair on transformation. Two mutations that cause a Uvr- and phenotype (uvr-1 and uvr-42) were shown to have strikingly different effects on repair of ultraviolet-irradiated transforming DNA. Genetic and kinetic evidence is presented to show that integrated DNA was apparently repaired by both excision and postreplication repair in wild-type and in uvr-1 recipients, although the latter excise pyrimidine dimers very slowly. In uvr-42 mutants, which are defective in incision at pyrimidine dimers, dimer-containing DNA was integrated. Postreplication repair apparently saved uvr-42 recipient cells from the lethal effects of integrated dimers, but the recombination events accompanying postreplication repair greatly reduced the linkage between closely linked genetic markers in the donor DNA. Repair of transforming DNA in a recG recipient, which does excision repair but not postreplication repair, was nearly as efficient as in wild-type cells. However, in this recipient linkage was altered only slightly, if at all, compared with wild-type cells. The apparent reduction in size of integrated regions of ultraviolet-irradiation transforming DNA probably results mainly from postreplication repair of larger integrated regions.     

DDB2-Independent Role for p53 in the Recovery from Ultraviolet Light-Induced Replication Arrest

Ultraviolet light (UV light) induces helix distorting DNA lesions that pose a block to replicative DNA polymerases. Recovery from this replication arrest is reportedly impaired in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) fibroblasts and primary fibroblasts lacking functional p53. These independent observations suggested that the involvement of p53 in the recovery from UV-induced replication arrest was related to its role in regulating the global genomic subpathway of NER (GG-NER). Using primary human fibroblasts, we confirm that the recovery from UV-induced replication arrest is impaired in cells lacking functional p53 and in primary XP fibroblasts derived from complementation groups A or C (XP-A and XP-C) that are defective in GG-NER. Surprisingly, DNA synthesis recovered normally in GG-NER-deficient XP complementation group E (XP-E) cells that carry mutations in the p53 regulated DNA repair gene DDB2 and are specifically defective in the repair of cyclobutane pyrimidine dimers (CPD) but not pyrimidine (6-4) pyrimidone photoproducts. Disruption of p53 in these XP-E fibroblasts prevented the recovery from UV-induced replication arrest. Therefore, the roles of p53 and GG-NER in the recovery from UV-induced replication are separable and DDB2-independent. These results further indicate that primary human fibroblasts expressing functional p53 efficiently replicate DNA containing CPD whereas p53-deficient cells do not, consistent with a role for p53 in permitting translesion DNA synthesis of these DNA lesions.     

Complementation analysis of xeroderma pigmentosum variants

DNA repair after UV exposure was studied in multinucleate cells, obtained after fusion of excision-defective and variant xeroderma pigmentosum fibroblasts. Optimal fusion conditions were determined, facilitating the measurement of DNA replication in heterokaryons. In unirradiated multikaryons, entry into the S phase was depressed, when compared with unfused cells. The extent of the depression of S phase entry was dependent on the fusion conditions. In heterokaryons obtained after fusion of XP variant (6 different strains) with excision-defective XP (three cell strains from complementation groups A, C and D) both unscheduled DNA synthesis and postreplication repair after UV irradiation were restored to normal levels. In contrast, complementation was not observed after pairwise fusion of the XP variant cell strains. These results suggest that the XP variants comprise a single complementation group, different from complementation groups A, C and D.     

Cytotoxicity and mutagenicity of aflatoxin dichloride in normal and repair deficient diploid human fibroblasts

The cytotoxic and mutagenic effect of aflatoxin B1-dichloride (AFB1-Cl2), a direct-acting carcinogen which is a model for the proposed ultimate reactive metabolite of AFB1 (the 2,3-epoxide), was compared in normal, repair-proficient, diploid human fibroblasts and in complementation Group A xeroderma pigmentosum cells (XP12BE) which are virtually incapable of excision repair of DNA damage induced by ultraviolet radiation, the 7,8-diol-9,10-epoxide of benzo[alpha]pyrene, and several reactive aromatic amide derivatives. The XP cells were significantly more sensitive than normal to the cytotoxic and mutagenic effects of AFB1-Cl2, not only as a function of concentration administered but also of the number of AFB1-Cl2 residues initially bound to DNA. Cytotoxicity was determined from survival of colony-forming ability; resistance to 6-thioguanine was the genetic marker used for mutagenicity. We compared the rate of loss of AFB1-Cl2-DNA adducts from cells treated and held in the non-dividing state (confluent) over several days, as well as their ability to recover from the potentially mutagenic and/or cytotoxic effects of the agent. AFB1-Cl2 residues were lost from both strains of cells and both exhibited a gradual increase in survival. However, the rate of loss of adducts from the DNA in the normal cells was more rapid than in XP cells and they exhibited recovery from higher doses of AFB1-Cl2 than XP cells. The major primary DNA adduct formed in the human cells and in isolated DNA was a chemically unstable guanine derivative which could undergo a change in structure with time posttreatment to form a more stable secondary adduct. The cytotoxic effect of AFB1-Cl2 was highly correlated with the presence of either of these guanine adducts. Evidence suggests that the primary adduct is an N7-guanine adduct. The kinetics of the loss of this guanine and its transformation into the more stable secondary adduct resembled that reported recently for the major primary DNA adduct formed by the reaction of AFB1 at the N-7 position of guanine in the DNA of normal and XP cells and its transformation into the putative AFB1-ring opened triamino pyrimidyl structure.     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Effects of a 6-fluoro substituent on the metabolism of benzo(a)pyrene 7,8-dihydrodiol to bay-region diol epoxides by rat liver enzymes

Metabolism of trans-7,8-dihydroxy-7,8-dihydro-6-fluorobenzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450c, has been examined. Although both the fluorinated and unfluorinated 7,8-dihydrodiol formed from benzo(a)pyrene by liver microsomes share (R,R)-absolute configuration, the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,5- and 9,10-dihydrodiols are also greater than 97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (7R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified monooxygenase system, this pathway accounts for 76-82% of the total metabolites from the 7,8-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-9,10-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,8-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.     

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.     

Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

Summary Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant.Irradiated cells from two mutants that display no excision capacity exhibit a gradual arrest of thymidine incorporation within 20 h after the initial decline. This arrest of incorporation is not observed in mutants exhibiting only partial defects in excision repair.Recovery of the ability to synthesize nascent DNA in long segments is normal in cells from the two mutants that display no excision capacity, indicating that recovery does not depend upon the excision of pyrimidine dimers from cellular DNA. Recovery of that ability is not observed, however, in cells from one partially excision-defective mutant, two of three postreplication repair-defective mutants, two of four mutants defective in both excision and postreplication repair, and one of five repair-proficient mutagen-sensitive mutants. These results indicate that recovery of normal DNA replication in irradiated Drosophila cells depends upon the activity of several functions.Abbreviation used UV ultraviolet light — principal wavelength 254 nm     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Postreplication repair of deoxyribonucleic acid and daughter strand exchange in uvr- mutants of Bacillus subtilis

The fate of pyrimidine dimers in deoxyribonucleic acid (DNA) newly synthesized by Bacillus subtilis after ultraviolet irradiation was monitored by use of a damage-specific endonuclease that introduces single-strand breaks adjacent to nearly all of the dimer sites. Two Uvr- strains, one defective in the initiation of dimer excision and the other defective in a function required for efficient dimer excision, were found to be similar to their wild-type parent in the kinetics and extent of converting low-molecular-weight DNA newly synthesized after ultraviolet irradiation to high molecular weight. In the Uvr- strains large molecules of newly synthesized DNA remained susceptible to nicking by the damage-specific endonuclease even after extended incubation in growth medium, whereas the enzyme-sensitive sites were rapidly removed from both preexisting and newly synthesized DNA in Uvr+ cells. Our results support the hypothesis that postreplication repair in bacteria includes recombination between dimer-containing parental DNA strands and newly synthesized strands.     

Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their DNA. 1969

Xeroderma pigmentosum (XP) is a recessively transmitted disorder of man characterized by increased sensitivity to ultraviolet light. Homozygous, affected individuals, upon exposure to sunlight, sustain severe damage to the skin; this damage is characteristically followed by multiple basal and squamous cell carcinomas and not uncommonly by other malignant neoplasia. A tissue culture cell line was derived from the skin of a man with XP. Our measurements of ultraviolet-induced pyrimidine dimers in cellular DNA show that normal diploid human skin fibroblasts excise up to 70 per cent of the dimers 24 hours, but that fibroblasts derived from the individual with XP excise less than 20 per cent in 48 hours. Alkaline gradient sedimentation experiments show that during the 24 hours after irradiation of normal cells a large number of single-stranded breaks appear and then disappear. Such changes are not observed in XP cells. XP cells apparently fail to start, the excision process because they lack the required function of an ultraviolet-specific endonuclease. These findings, plus earlier ones of Cleaver on the lack of repair replication in XP cells, raise the possibility that unexcised pyrimidine dimers can be implicated in the oncogenicity of ultraviolet radiation.     

A further definition of characteristics of DNA-excision repair in xeroderma pigmentosum complementation group A strains

The location in the genome of excision repair following exposure to UV (254 nm) of two XP complementation group A strains, XP12BE and XP8LO, that differ considerably in their excision-repair rates, have been determined. Capacity for repair in XP8LO has also been determined. Sites repaired in DNA in a 24-h post-UV period were located relative to the remaining pyrimidine dimers using the M. luteus UV-endonuclease to nick partially repaired DNA and sedimentation in alkaline sucrose to size the resulting DNA. Repair in group A occurs randomly throughout the genome in a manner similar to that observed for normal cells but in contrast to domain-limited repair in group C strains. This observation defines a further similarity of the excision repair detected in group A compared to normal cells that is in addition to the previously reported related characteristics of the respective excision rate curves. A reduced repair capacity in XP8LO relative to normal cells was detected. This strain, which repairs DNA at an initial rate identical to that of normal strains when irradiated with doses of 5 J/m2 or less, repairs DNA at a slower than normal but constant rate at higher doses. This leads to the suggestion that XP8LO is defective in the number of repair enzyme complexes compared to normal cells.     

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.     

Transient appearance of photolyase-induced break-sensitive sites in the DNA of ultraviolet light-irradiated Syrian hamster fetal cells

Syrian hamster fetal fibroblasts (HFC) were examined for photolyase-induced break-sensitive sites after ultraviolet light (UV) exposure and growth. These sites, observed in excision-defective human xeroderma pigmentosum (XP) cells, are due to cleavage of the internal phosphodiester bond of UV-induced pyrimidine dimers. Excision-inefficient HFC acquired photolyase-induced break-sensitive sites during incubation after UV (10 J/m2). However, these were observed transiently, with a maximum of 5% of the pyrimidine dimers at 9 h post UV; by 18 h they were undetectable. Caffeine (1 mM) delayed the peak of photolyase-induced break-sensitive sites by 2 h. In human XP cells photolyase-induced break-sensitive sites accumulate to a plateau level of about 20% of the pyrimidine dimers. The present results extend to rodent cells the observation that cleavage of the internal phosphodiester bond of pyrimidine dimers may be an early step in their excision repair. Furthermore, the data suggest that photolyase-induced break-sensitive sites might be necessary for replication bypass at pyrimidine dimers.