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.     

Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.     

Excision repair characteristics of denV-transformed xeroderma pigmentosum cells

Introduction of the denV gene of phage T4, encoding the pyrimidine dimer-specific endonuclease V, into xeroderma pigmentosum cells XP12RO(M1) was reported to result in partial restoration of colony-forming ability and excision repair synthesis. We have further characterized 3 denV-transformed XP clones in terms of rates of excision of pyrimidine dimers and size of the resulting resynthesized regions following exposure to 100 J/m2 from an FS-40 sunlamp. In the denV-transformed XP cells we observed 50% dimer removal within 3-6 h after UV exposure as compared to no measurable removal in the XP12RO(M1) line and 50% dimer excision after 18 h in the GM637A human, control cells. Dimer removal was assayed with Micrococcus luteus UV-endonuclease in conjunction with sedimentation of treated DNA in alkaline sucrose gradients. The size of the resulting repaired regions was determined by the bromouracil photolysis technique. Based on the photolytic sensitivity of DNA repaired in the presence of bromodeoxyuridine, we calculated that the excision of a dimer in the GM637A cells appears to be accompanied by the resynthesis of a region approximately 95 nucleotides in length. Conversely, the resynthesized regions in the denV-transformed clones were considerably smaller and were estimated to be between 13 and 18 nucleotides in length. These results may indicate that either the endonuclease that initiated dimer repair dictated the size of the resynthesized region or that the long-patch repair observed in the normal cells resulted from the repair of non-dimer DNA lesions.     

Low-level DNA exchanges in normal human and xeroderma pigmentosum cells after UV irradiation

We have used a T4 endonuclease V assay method for UV-induced pryrimidine dimers in cellular DNA in vivo to obtain evidence for recombinational DNA exchanges after UV irradiation of normal human and Xeroderma pigmentosum (XP) cells. Our data indicate that the endonuclease-sensitive sites in excision-defective XP cells are removed very slowly from the irradiated parental strands and appear concomitantly in daughter strands newly synthesized during post-UV incubation. In the defective XP cells, the extent of appearance of sensitive sites in daughter strands synthesized during a period of 24 h after 10 J/m² appears to be small, probably less than 15% of the initial number of sensitive sites detected in cellular parental strands. Demonstration of such exchanges between normal-density parental and 5-bromodeoxyuridine-labeled daughter strands by alkaline CsCl isopycnic centrifugation was unsuccessful. Further, the extent is much lower in normal human cell because of their efficiet excision repair of the dimers before and after exchanges than in the defective XP cells.     

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.     

The relationship between pyrimidine dimers and replicating DNA in UV-irradiated human fibroblasts.

The relationship between pyrimidine dimers (measured as endonuclease-sensitive sites) and newly-synthesized DNA has been examined in several different ways, with the following results:- 1. After UV-irradiation of normal human fibroblasts the frequency of pyrimidine dimer sites in sections of DNA which have been synthesized immediately before the UV-irradiation is similar to that in the bulk DNA. 2. The frequency of pyrimidine dimer sites in the parental strands of replicating DNA in UV-irradiated normal human fibroblasts is similar to that in the bulk DNA. 3. In UV-irradiated XP variant cells the size of DNA synthesized in the presence of caffeine immediately after UV irradiation accurately corresponds with the average interdimer distance in the parental DNA. This suggests that in this experimental situation each pyrimidine dimer gives rise to a disocntinuity or a termination site in the daughter strand.     

DNA Repair in Potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times (~ 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure ~ 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though ~ 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.     

Effect of Caffeine on Postreplication Repair in Human Cells

DNA synthesized shortly after ultraviolet (UV) irradiation of human cells is made in segments that are smaller than normal, but at long times after irradiation the segments made are normal in size. Upon incubation, both the shorter and the normal segments are elongated and joined by the insertion of exogenous nucleotides to form high molecular weight DNA as in nonirradiated cells. These processes occur in normal human cells, where UV-induced pyrimidine dimers are excised, as well as in xeroderma pigmentosum (XP) cells, where dimers are not excised. The effect of caffeine on these processes was determined for both normal human and XP cells. Caffeine, which binds to denatured regions of DNA, inhibited DNA chain elongation and joining in irradiated XP cells but not in irradiated normal human or nonirradiated cells. Caffeine also caused an alteration in the ability to recover synthesis of DNA of normal size at long times after irradiation in XP cells but not in normal cells.     

Recovery of the Ability to Synthesize DNA in Segments of Normal Size at Long Times After Ultraviolet Irradiation of Human Cells

DNA synthesized in human cells within the first hour after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in nonirradiated cells. The size of these segments approximates the average distance between pyrimidine dimers in the parental DNA. This suggests that the dimers interrupt normal DNA synthesis and result in gaps in the newly synthesized DNA. However, DNA synthesized in human cells at long times after irradiation is made in segments equal or nearly equal to those synthesized by nonirradiated cells. The recovery of the ability to synthesize DNA in segments of normal size occurs in normal human cells, where the dimers are excised, and also in cells of the human mutants xeroderma pigmentosum (XP), where the dimers remain in the DNA. This observation implies that the pyrimidine dimer may not be the lesion that causes DNA to be synthesized in smaller than normal segments.     

A sensitive, enzymatic assay for the detection of closely opposed cyclobutyl pyrimidine dimers induced in human diploid fibroblasts

A sensitive, enzymatic assay has been developed for the detection of closely opposed cyclobutyl pyrimidine dimers induced in UV-irradiated human diploid fibroblasts. In this assay closely opposed dimers are detected as bifilar enzyme-sensitive sites. Single-strand incisions are made at the positions of individual pyrimidine dimers through the action of M. luteus pyrimidine dimer-DNA glycosylase. Incisions at closely opposed dimers, effectively expressed as double-strand breaks, are quantified from the resulting reduction in DNA double-strand molecular weight as determined by velocity sedimentation through neutral sucrose density gradients. The stability of the bacteriophage lambda cos site under our reaction conditions indicates that opposed incisions must be relatively close to be expressed as a double-strand break. The dose response for the induction of bifilar enzyme-sensitive sites in mammalian cells was found to be complex but can be approximated by a function that increases as the 1.2-1.4 power of UV dose. The frequency of bifilar enzyme-sensitive sites observed decreased during postirradiation incubation of excision-repair-proficient human diploid fibroblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m2 (254 nm). By comparison, over 80% of the bifilar enzyme-sensitive sites induced in fibroblasts assigned to xeroderma pigmentosum complementation group A remained detectable 24 h after irradiation. The implications of these results for models addressing the induction and repair of closely opposed pyrimidine dimers are discussed.     

Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.     

Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.

A UV-resistant revertant (XP129) of a xeroderma pigmentosum group A cell line has been reported to be totally deficient in repair of cyclobutane pyrimidine dimers (CPDs) but proficient in repair of 6-4 photoproducts. This finding has been interpreted to mean that CPDs play no role in cell killing by UV. We have analyzed the fine structure of repair of CPDs in the dihydrofolate reductase gene in the revertant. In this essential, active gene, we observe that repair of the transcribed strand is as efficient as that in normal, repair-proficient human cells, but repair of the nontranscribed strand is not. Within 4 h after UV at 7.5 J/m2, over 50% of the CPDs were removed, and by 8 h, 80% of the CPDs were removed. In contrast, there was essentially no removal from the nontranscribed strand even by 24 h. Our results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs from the transcribed strands of expressed genes is essential for UV resistance.     

Sequence effect on incision by (A)BC excinuclease of 4NQO adducts and UV photoproducts.

Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer. The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light. Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine. The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct. By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning. In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75%. Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50%. The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer.     

Excision repair of ultraviolet damage in mammalian cells. Evidence for two steps in the excision of pyrimidine dimers

The incidence of pyrimidine dimer formation and the kinetics of DNA repair in African green monkey kidney CV-1 cells after ultraviolet (UV) irradiation were studied by measuring survival, T4 endonuclease V-sensitive sites, the fraction of pyrimidine dimers in acid-insoluble DNA as determined by thin layer chromatography (TLC), and repair replication. CV-1 cells exhibit a survival curve with extrapolation number n = 7.8 and Do = 2.5 J/m2. Pyrimidine dimers were lost from acid-insoluble DNA more slowly than endonuclease-sensitive sites were lost from or new bases were incorporated into high molecular weight DNA during the course of repair. Growth of CV-1 cultures in [3H]thymidine or X-irradiation (2 or 10 krads) 24 h before UV irradiation had no effect on repair replication induced by 25 J/m2 of UV. These results suggest that pyrimidine dimer excision measurements by TLC are probably unaffected by radiation from high levels of incorporated radionuclides. The endonuclease-sensitive site and TLC measurements can be reconciled by the assumption that pyrimidine dimers are excised from high molecular weight DNA in acid-insoluble oligonucleotides that are slowly degraded to acid-soluble fragments.     

Use of UV endonuclease from Micrococcus luteus to monitor the progress of DNA repair in UV-irradiated human cells

A sensitive enzymatic assay has been developed to follow the progress of NDA repair in human cells exposed to UV radiation. The assay employs an endonuclease selectively active at sites containing pyrimidine dimers in UV-damaged DNA. Primary fibroblasts are exposed to 254 nm radiation and incubated for specified times, their radioactivity labelled DNA is isolated and treated with a UV endonuclease extensively purified from Micrococcus luteus. Endonuclease-susceptible site remaining in the DNA are subsequently observed as single-strand scissions by sedimentation in alkaline sucrose gradients. In comparison to the situation with excision-proficient normal cells, those derived from patients suffering from either the classical or the De Sanctis-Cacchione clinical form of Xeroderma pigmentosum (XP) exhibit a marked diminution in the rate of disappearance of nuclease-susceptible lesions with time of post-UV incubation.     

A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region

The uvrA, uvrB, and uvrC proteins of Escherichia coli were purified from strains that greatly overproduce these proteins. Using the purified proteins, the UVRABC nuclease was reconstituted in vitro. The reconstituted enzyme acted specifically on DNA damaged with UV, cis-platinum, and psoralen plus near UV. When UV-irradiated DNA was used as substrate, the enzyme made two cuts on the damaged DNA strand, one on each side of the damaged region. The enzyme hydrolyzed the eighth phosphodiester bond on the 5′ side of pyrimidine dimers. On the 3′ side of pyrimidine dimers, the UVRABC nuclease cut the fourth or the fifth phosphodiester bond 3′ to pyrimidine dimers. The oligonucleotide with the damaged bases that is generated by these two cuts was released during treatment with the enzyme. We have also obtained evidence suggesting that the enzyme acts by the same mechanism on PydC photoproducts which are thought to be of primary importance in UV-induced mutagenesis.     

DNA sequence dependence of closely opposed cyclobutyl pyrimidine dimers induced by UV radiation

Treatment of UV-irradiated DNAs with Micrococcus luteus pyrimidine dimer-DNA glycosylase results in the formation of double-strand breaks due to cleavage at closely opposed pyrimidine dimers. To determine if the induction of closely opposed dimers is significantly affected by DNA nucleotide sequence, end-labeled DNA fragments of known nucleotide sequence were UV irradiated, incubated with pyrimidine dimer-DNA glycosylase, and analyzed by electrophoresis through nondenaturing polyacrylamide gels. Distinct bands of increased electrophoretic mobility were observed, indicating that bifilar cleavage had occurred with greater probability at specific sites in each DNA sequence. In vitro enzymatic photoreactivation of dimers prior to treatment with pyrimidine dimer-DNA glycosylase prevented the appearance of bands. DNA sequence analysis revealed the presence of closely opposed runs of pyrimidines at sites of more frequent bifilar cleavage. Our results indicate that the induction of closely opposed dimers occurs with greater probability at specific sites in DNA sequences and that such sites are characterized by the presence of closely opposed pyrimidine runs.     

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.