UV-irradiated Human Cells Research Articles

Identification of a large genomic region in UV-irradiated human cells which has fewer cyclobutane pyrimidine dimers than most genomic regions.

Size separation after UV-endonuclease digestion of DNA from UV-irradiated human cells using denaturing conditions fractionates the genome based on cyclobutane pyrimidine dimer content. We have examined the largest molecules available (50-80 kb; about 5% of the DNA) after fractionation and those of average size (5-15 kb) for content of some specific genes. We find that the largest molecules are not a representative sampling of the genome. Three contiguous genes located in a G+C-rich isochore (tyrosine hydroxylase, insulin, insulin-like growth factor II) have concentrations two to three times greater in the largest molecules. This shows that this genomic region has fewer pyrimidine dimers than most other genomic regions. In contrast, the beta-actin genomic region, which has a similar G+C content, has an equal concentration in both fractions as do the p53 and beta-globin genomic regions, which are A+T-rich. These data show that DNA damage in the form of cyclobutane pyrimidine dimers occurs with different probabilities in specific isochores. Part of the reason may be the relative G+C content, but other factors must play a significant role. We also report that the transcriptionally inactive insulin region is repaired at the genome-overall rate in normal cells and is not repaired in xeroderma pigmentosum complementation group C cells.

Preferential repair of the transcribed DNA strand in the dihydrofolate reductase gene throughout the cell cycle in UV-irradiated human cells

We examined repair of UV-induced cyclobutane pyrimidine dimers (CPD) in each strand of the expressed dihydrofolate reductase gene in human cells in different phases of the cell cycle: G1, early S, middle S, late S, and G2/M. After 4 h of incubation, repair of the transcribed strand was substantially more efficient than repair of the non-transcribed strand in all phases. Furthermore, we observed no remarkable cell cycle-dependent differences in either the initial lesion frequency or the efficiency of repair of the transcribed strand. We conclude that transcription coupled repair operates generally and with high efficiency throughout the cell cycle.

Amplification of Epstein-Barr virus-based shuttle vectors by ultraviolet light in human cells

In order to approach the mechanism of gene amplification, we have developed a model system in human cells based on the use of episomally-replicating shuttle vectors. Shuttle vectors carrying the replication origin of the Epstein-Barr virus can be stably maintained in human cells. These vectors replicate as an episome with a low copy number. We also constructed hybrid plasmids containing both the EBV and the SV40 replication origins. These molecules are able to replicate episomally either like an EBV vector or like SV40 if the SV40 large T antigen is provided at the same time. UV irradiation of both human adenovirus transformed 293 or SV40-transformed MRC5 host cells leads to vector amplification whatever the type of replication origin used for the episomal maintenance. Our result clearly shows that the EBV latent replication origin (OriP), in the presence of the Epstein-Barr nuclear antigen-1 (EBNA-1) and the SV40 large T antigen, is sensitive to over-replication in UV-irradiated human cells. Since the UV doses were small enough to induce very little damage, if any, on the plasmid sequences, this amplification should be mediated through a cellular factor acting in trans. The interest in using shuttle vectors for this kind of study lays in the easy analysis of the amplified vectors in rescued bacterial colonies. The accuracy of the amplification process can be monitored by studying restriction maps of individual plasmid molecules or more precisely the integrity of a target gene, such as the lacZ′ sequence, carried by our vectors.

DNA repair within nucleosome cores of UV-irradiated human cells

We have compared the distributions of repair synthesis and pyrimidine dimers (PD) in nucleosome core DNA during the early (fast) repair phase and the late (slow) repair phase of UV-irradiated human fibroblasts. As shown previously [Lan, S. Y., & Smerdon, M. J. (1985) Biochemistry 24, 7771-7783], repair synthesis is nonuniform in nucleosome core particles during the fast repair phase, and the distribution curve can be approximated by a model where repair synthesis occurs preferentially in the 5' and 3' end regions. In this report, we show that, during the slow repair phase, [3H]dThd-labeled repair patches are much more uniformly distributed in core DNA, although they appear to be preferentially located in sequences degraded slowly by exonuclease III. This change in distribution cannot be explained by an increase in patch size during slow repair, since the size of these patches actually decreases to about half the size measured during the fast repair phase. Furthermore, PD mapping within core DNA at the single-nucleotide level demonstrated that, at least within the 30-130-base region from the 5' end, there is little (or no) selective removal of PD during the fast repair phase. However, the nonuniform distribution of repair synthesis obtained during fast repair throughout most of the core DNA region (approximately 40-146 bases) is accounted for by the nonuniform distribution of PD in core DNA. The near-uniform distribution of repair synthesis observed during slow repair may result from more extensive nucleosome rearrangement and/or nucleosome modification during this phase.

Rapid repair kinetics of pyrimidine(6–4)pyrimidone photoproducts in human cells are due to excision rather than conformational change

UV-induced pyrimidine(6-4)pyrimidone photoproducts in DNA of mammalian cells are apparently repaired much more rapidly than cyclobutane dimers. Since only immunological assays for (6-4) photoproducts have been sensitive enough for repair measurements, it was possible that these apparently rapid repair kinetics reflected a change in physical conformation of antibody-binding sites, resulting in epitope loss rather than excision. To discriminate between these possibilities, we developed a procedure to photochemically convert (6-4) photoproducts to single-strand breaks in UV-irradiated DNA with a background low enough to permit repair measurements. Analysis of a specific DNA sequence indicated that photoinduced alkali-labile sites (PALS) were induced with the same site-specificity as (6-4) photoproducts. Normal human and xeroderma pigmentosum (XP) variant cells rapidly excised (6-4) photoproducts measured as PALS, but little repair was seen in cells from XP complementation group A. These repair kinetics corresponded to those determined in the same samples by radioimmunoassay of (6-4) photoproducts. Thus we conclude that the rapid repair of (6-4) photoproducts observed in UV-irradiated human cells is not the result of a conformational change resulting in epitope loss, but reflects excision of this photoproduct from DNA.

Some studies on the DNA-repair-eliciting and genotoxic activity of cell-free extracts of Lactobacillus bifidus

Cell-free extracts of Lactobacillus bifidus have been reported as possessing DNA-repair-eliciting properties in UV-irradiated human cells, and suggestions have been made that these extracts could be used to protect human skin cells from DNA-damaging effects induced by natural UV light. In view of the importance of these findings, and because extracts of other bifidobacteriae had previously been shown to possess genotoxic activity in bacterial systems, it seemed appropriate to perform some experiments aimed at evaluating the ability of cell-free extracts of L. bifidus, as well as the bacterial suspension medium, to modulate DNA repair and/or to exert potentially adverse genotoxic effects in a variety of mammalian cells in culture. Chinese hamster cells, human fibroblasts, and human lymphocytes were used to evaluate the influence of the extract on the repair of UV-damaged DNA and on several biological effect (cell cycle progression, cell killing, chromosomal aberrations, and sister-chromatid exchanges) induced by DNA-damaging agents. The results show that neither the extract nor the suspension have any influence on DNA repair or other biological endpoints induced by UV and other DNA-damaging agents. In conclusion, the present findings do not indicate that cell-free extracts of L. bifidus specifically promote the repair of UV-damaged DNA in human cells. Neither do they indicate that these extracts have a promoting activity on UV-induced (misrepair) mutagenesis in mammalian cells. Finally, the present experiments indicate that the L. bifidus extracts used in our experiments are devoid of any direct mutagenic and/or genotoxic activity in mammalian cells.

(6–4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers

A polyclonal antiserum raised against UV-irradiated DNA can be used to assay cyclobutane pyrimidine dimers and Pyr(6-4)Pyo photoproducts specifically by changing the nature of the 32P-labelled antigen. Pyr(6-4)Pyo photoproducts were removed faster than cyclobutane dimers in UV-irradiated human, hamster and mouse cells. Xeroderma pigmentosum cells from complementation groups A, C and D were deficient in the repair of both lesions.

Initial rates of DNA incision in UV-irradiated human cells differences between normal, xeroderma pigmentosum and tumour cells

Following UV-irradiation and in the presence of inhibitors of DNA synthesis (hydroxyurea and 1-β- d-arabinofuranosylcytosine) human cells accumulate strand breaks in their DNA—as a result of enzymic incision without subsequent rejoining. We have developed a sensitive procedure which makes stringent use of these inhibitors so as to maximize the frequency of breaks detected after low levels of UV (0.25–10 Jm −2 and to permit analysis of the kinetics of break accumulation over short intervals after irradiation (up to 90 min). Since the rate of accumulation of breaks declines quickly with time of incubation (not simply as a consequence of substrate depletion), we have calculated initial rate constants by extrapolating to zero time for a range of UV doses (i.e. different substrate concentrations). Using these constants as indices of enzymic incision, we have compared a wide range of human cell types, and have (in some cases) been able to estimate the enzymatic parameters K M and V max for the incision step. Assessed in this way the human cells tested fall into a number of distinct categories. Fibroblasts from normal embryos and from xeroderma pigmentosum (XP) variant and Bloom's syndrome show high and uniform levels of incision readily distinguishable from XP(A), in turn distinct from XP(D). Tumour-derived cells and SV40-transformed fibroblasts also fall into a group with similar incision capacity, significantly lower than that of normal diploid cells. We discuss possible reasons for this distinction, and evaluate the use of inhibitors in repair studies. One of the key early steps in the repair of DNA in UV-irradiated cells is enzymic incision at damage sites. There is a well-defined group of human genetic diseases (xeroderma pigmentosum, XP) associated with high risk of malignancy in which, in all but one complementation group, the defect is located at or around incision (Robbins et al., 1974; Setlow, 1978). In bacteria incision appears to be a major site of regulation of excision repair (Grossman, 1974), and we believe that enzymatic analysis of incision in eukaryote cells, too, will provide information about the molecular nature and the control of this process. In UV-irradiated human cells, incision is readily detected by incubation with DNA synthesis inhibitors such as hydroxyurea (HU) and/or 1-β- d-arabinofurasylcytosine (araC); repair DNA synthesis is blocked, and the breaks introduced in the DNA by cellular endonucleases, which would otherwise be resealed after repair, accumulate within minutes of irradiation. This technique for studying the cellular response to UV has yielded valuable information about the capacity of various mammalian cell lines to repair DNA damage (Collins, 1977; Hiss and Preston, 1977; Collins and Johnson, 1979; Dunn and Regan, 1979; Erixon and Ahnström, 1979), and now we apply it in a quantitative comparison of different human cell lines. These include cells from normal individuals and from patients with diseases associated with DNA repair defects, and also tumour-derived and virally transformed cells. We find that different cell types fall into well defined classes according to initial apparent incision activity.

Repair Replication Schemes in Bacteria and Human Cells

The chapter discusses the schemes that remove damage from the DNA and attempt restoration of the original nucleotide sequence. The excision-repair of damaged DNA must involve a coordinated sequence of steps beginning with recognition of the lesion. After phosphodiester bonds are broken, the integrity of the interrupted DNA strand is restored through the enzymic processes of degradation, repair resynthesis, and ligation. Pathways for excision-repair in E.coli are elucidated through analysis of mutants deficient in various enzyme activities. A pathway mediated by DNA polymerase I that results in short repair patches approximately 20 nucleotides in extent; a pathway that results in a somewhat larger average patch size, probably due to intrinsic properties of DNA polymerases II and III; and a pathway that is induced in response to DNA damage and is dependent upon protein synthesis, requires recA+ and lexA+, and leads to much longer patches. Short patches are initiated and completed earlier than the long patches produced by the inducible pathway. In UV-irradiated human cells, only a single class of repair patches comparable in size to the short patches in E.coli, is synthesized at both early and late times after irradiation. The same patch size distribution also results from the treatment of human cells by angelicin or 8-methoxypsoralen plus long-wavelength UV, or by activated aflatoxin B1.

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [(3)H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The (3)H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg(2+)-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.

Photoreactivation of thymine dimers in UV-irradiated human cells: Unique dependence on culture conditions

UV-irradiated human fibroblasts in tissue culture were exposed to photoreactivating light in an attempt to demonstrate a light-dependent loss of thymine dimers from the acid-insoluble fraction of the DNA. The only experimental conditions in which this phenomenon was observed was if the cells were grown for at least 10 days in Dulbecco's modified Eagle's minimum essential medium. Such cells lost a maximum of between 10–30% of the thymine dimers from their DNA during illumination for 1 h. When cells were grown in a variety of other media the phenomenon was not observed. The present experiments do not discriminate between true enzymatic photoreactivation and a medium-dependent photosensitization phenomenon that is not enzymatic in nature.

The inhibition of repair in UV irradiated human cells

We have used three different assay procedures to determine the effects of hydroxyurea on excision repair in UV-irradiated HeLa cells. The results were as follows. (a) At the cytological level, incubation of UV-irradiated metaphase cells with hydroxyurea caused chromosome decondensation. (b) Using a modified alkaline sucrose gradient sedimentation technique involving minimal lysis before centrifugation, we found a marked retardation in the sedimentation of DNA from UV-irradiated cells incubated for a short period with hydroxyurea. (c) The effect of hydroxyurea on the incorporation of[3H]thymidine by UV-irradiated G1 cells was found to depend on the concentration of thymidine present in the medium. Normal primary human cells resemble HeLa cells in the response of chromosomes and DNA to UV plus hydroxyurea. Xeroderma pigmentosum cells, deficient in excision repair, are not sensitive to hydroxyurea in our assays. Chromosome decondensation and retarded DNA sedimentation occur also after incubation of irradiated HeLa cells with deoxyadenosine, but not thymidine, at concentrations which inhibit semiconservative DNA synthesis. The effects of hydroxyurea or deoxyadenosine on chromosomes and DNA are not seen if all four deoxyribonucleoside precursors of DNA are supplied exogenously. These results point to an inhibition of repair DNA synthesis by hydroxyurea (or deoxyadenosine), at the level of the supply of DNA precursors, i.e. in the same way that these agents inhibit semiconservative DNA synthesis. In the presence of these inhibitors, single-strand gaps accumulate in the DNA.

Use of enzymatic assay to evaluate UV-induced DNA repair in human and embryonic chick fibroblasts and multinucleate heterokaryons derived from both.

A sensitive enzymatic assay has been utilized to monitor repair of UV-induced damage to DNA in primary human and embryonic chick cells and in multinucleate heterokaryons artificially derived from both. The assay exploits the unique ability of a purified repair endonuclease to attack UV-irradiated DNA at sites containing pyrimidine dimers. These nuclease-susceptible sites are subsequently observed as single-strand scissions by velocity sedimentation in alkaline sucrose gradients. Incubation of UV-damaged cultures followed by extraction and enzymatic analysis of the radioactively labeled DNA enables one to trace the disappearance of such sites in vivo and hence to monitor endogenous repair activity. When UV-irradiated human cells are incubated in the dark, the curve for site removal exhibits a two-phase exponetial decline; i.e. there exists a fast component responsible for elimination of 60% of the initial damage and a second one approximately 7 times slower in rate. The removal of sites is not further enhanced by exposing cells to blacklight during post-UV incubation. Conversely, UV-damaged chick cells rid their DNA of all nuclease-susceptible sites rapidly (i.e. at an exponential rate approximately 13 times faster than the fast component of site removal in human cells) when incubated under blacklight but not when kept in the dark. These data indicate the presence in human and embryonic chick cells of distinct enzymatic mechanisms for the elimination of dimer-containing sites. Wheneras human fibroblasts rely heavily on a light-independent process, excision-repair, chick fibroblasts possess a light-dependent mechanism, presumably photoenzymatic repair. Advantage has been taken of the contrasting repair properties of the human and embryonic chick fibroblasts to evaluate the extent to which each can assist the other in the removal of UV-induced damage from its DNA. The two cell types were fused to form giant human/chick heterokaryons containing a number of intact nuclei from both strains. Experimental conditions were selected so that UV-induced damage resided only in DNA foreign to the repair enzymes under study. Our results strongly suggest that repair enzyme(s) coded for by either fusion partner can remove dimer-containing sites from the DNA of the other with an efficiency comparable to that attained when acting on its own DNA in unfused, parental cells. Further, the light-requiring repair process supplied by the chick is more proficient at operating on these sites in human DNA than is excision-repair, the parallel mechanism available to human cells for this purpose.

Synthesis by UV-irradiated human cells of normal-sized DNA at long times after irradiation.

Although the DNA synthesized in human cells within the first hour after irradiation is in smaller segments than in unirradiated cells, the segments made at long times after irradiation are of normal size even though there has been no dimer excision. (auth)

Use of a 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.

Repair replication in human cells studied by sodium lodide isopycnic centrifugation of DNA in a fixed-angle rotor

A sensitive and quantitative method is described for direct measurement of repair replication in uv-irradiated human cells in tissue culture. The method is based on isopycnic centrifugation of repaired DNA in sodium iodide gradients in fixed angle rotors and determination of the amount of DNA by the fluorescence of a DNA ethidium bromide complex.