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.     

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.     

Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [(3)H]thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rates of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins.Our observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair.     

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.     

Cytological evidence for DNA chain elongation after UV irradiation in the S phase

Human cells irradiated with UV light synthesize lower molecular weight DNA than unirradiated cells. This reduction in molecular weight is greater in xeroderma pigmentosum (XP) cells than in normal cells. The molecular weight of DNA is further reduced by the addition of caffeine to XP cells. By several hours after irradiation, DNA fragments are barely detectable. Cells from excision-proficient and excision-deficient XP patients were studied autoradiographically to produce cytological evidence of DNA chain elongation. Replicate cultures with and without caffeine were synchronized and irradiated with UV light during the S phase. Caffeine was removed in G₂, and the cells were labeled with ³H-thymidine. Results showed significantly increased labeling during G₂ of excision-deficient XP cells. Labeling was dependent on both time of irradiation and presence of caffeine. The XP variant cells had no increase in labeling for any irradiation time.Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 1608. Supported by N.I.C. Grant TO1CA05170-10.     

Repair of DNA damage in shuttle vectors, virus, and chromosomal DNAs may depend on their biological imprinting--a 'Pygmalion' effect

We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their 'cultural background', i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.     

Repair of DNA damage in shuttle vectors,virus, and chromosomal DNAs may depend on their biological imprinting — a ‘Pygmalion’ effect

We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their ‘cultural background’, i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.     

Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells.

Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobuytl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis-syn thymine-thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts.     

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.     

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.     

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.     

Caffeine and human DNA metabolism: the magic and the mystery

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.     

Replication of DNA in UV-irradiatedEscherichia coli in the absence of amino acids

Since pyrimidine dimers are considered to be the cause of the synthesis of short DNA segments, normalization of DNA replication after UV irradiation should be in a temporal correlation with their removal. This correlation holds in exponentially growing excision-proficientEscherichia coli cells. However, when these cells are preincubated and postincubated without amino acids, synthesis of short segments continues although dimers are efficiently excised.     

The early and late modes of DNA replication in ultraviolet irradiated Syrian hamster embryo cells.

The nature of DNA replication in UV irradiated Syrian hamster embryo cells (HEC) was investigated by measuring the size distribution of nascent daughter strand DNA. During the early mode nascent strands are made in smaller pieces than in nonirradiated cells. The late mode begins when nascent strands recover to normal size. This was observed in HEC 5 h post-UV. When the late mode is operational, nascent strands elongate to parental size in greater than 2 h, whereas less than 3 h are required during early mode function. Evidence from split dose experiments demonstrates that the recovery of the size of nascent strands is not due to enhanced gap filling. Furthermore, pyrimidine dimers are probably recognized differently by the replication complex during early and late mode DNA synthesis. The late mode of replication could account for the ability of HEC to survive UV irradiation even though they are inefficient in both excision and postreplication repair.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

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.     

DNA Chain Elongation and Joining in Normal Human and Xeroderma Pigmentosum Cells after Ultraviolet Irradiation

DNA synthesized in human cells after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in unirradiated cells. Within several hours after irradiation these smaller units are both elongated and joined together. This repair process has been observed in normal human fibroblasts, HeLa cells, and fibroblasts derived from three types of xeroderma pigmentosum patients—uncomplicated with respect to neurological problems, complicated (de Sanctis-Cacchione syndrome), and one with the clinical symptoms of xeroderma pigmentosum but with normal repair replication. The ability of human cells to elongate and to join DNA strands despite the presence of pyrimidine dimers enables them to divide without excising the dimers present in their DNA. It may be this mechanism which enables xeroderma pigmentosum cells to tolerate small doses of UV radiation.     

Deoxyribonucleic acid excision repair in chromatin after ultraviolet irradiation of human fibroblasts in culture.

We have exposed confluent normal human fibroblasts to ultraviolet (UV) fluences of 5, 14, or 40 J/m2 and monitored the specific activity of post-UV repair synthesis in chromatin with [3H]thymidine pulses. We have shown that under conditions where no semiconservative deoxyribonucleic acid (DNA) synthesis is detectable, the specific activity of repair label in micrococcal nuclease resistant (core particle) DNA is about one-fifth that in bulk DNA at all three UV fluences. On the other hand, the distribution of thymine-containing pyrimidine dimers in bulk and nuclease-resistant regions measured either immediately after irradiation or at later times showed no significant differences; preferential labeling of linker (nuclease-sensitive) DNA during repair synthesis is thus apparently not due to a predominance of UV-induced photoproducts in linker relative to core particle DNA in the nucleosome. Pulse and pulse--chase experiments at 14 or 40 J/m2 with normal human or repair-deficient xeroderma pigmentosum (XP) cells showed that at most 30% of repair label in all these cells shifts from nuclease-sensitive (linker) DNA to nuclease-resistant (core particle) DNA.     

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.     

Persistence of pyrimidine dimers during post-replication repair in ultraviolet light-irradiated Escherichia coli K12

We have used a new assay for pyrimidine dimers to obtain evidence regarding the mechanism of post-replication repair of ultraviolet light-induced damage in excision-deficient (uvr) mutants of Escherichia coli. Our data indicate that dimers are gradually removed from the irradiated DNA under conditions permitting post-replication repair. Concomitantly, dimers appear in daughter strands synthesized after irradiation. The daughter strands initially contain gaps. During post-replication repair the gaps are filled and the originally discontinuous DNA is joined into long molecules resembling those observed in unirradiated control cells. Density transfer experiments reported by other investigators have provided evidence that the gap-filling involves exchanges between irradiated parental DNA and unirradiated daughter strands. The results of our experiments are in accord with this possibility and suggest that some dimers are included in the exchanged regions. Our data imply that intact, dimer-free DNA molecules are not necessarily generated by gap-filling and may not appear in uvr cells until several hours after u.v. irradiation. Instead, dimers may be gradually diluted among successive generations of DNA molecules synthesized after irradiation.