SATURATION OF DNA REPAIR IN MAMMALIAN CELLS*

Abstract—Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20J/m². We measured excision repair in normal human fibroblasts up to 80J/m². The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus , and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24 h following two 20J/m² doses separated by 24 h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the, rate limiting steps in repair.     

PYRIMIDINE DIMER SITES ASSOCIATED WITH THE DAUGHTER DNA STRANDS IN UV-IRRADIATED HUMAN FIBROBLASTS

Abstract. Pyrimidine dimer sites associated with the newly-synthesized DNA were detected during post-replication repair of DNA in UV-irradiated human fibroblasts. These pyrimidine dimer sites were inferred from a decrease in the molecular weight of pulse-labelled DNA after treatment with an extract of Micrococcus luteus containing UV-specific endonuclease activity. In DNA synthesized immediately after irradiation the frequency of these daughter strand dimer sites was 7–20% of that in the parental DNA. Such sites were found in fibroblasts from normal donors and from xeroderma pigmentosum patients (with defects in excision-repair or post-replication repair). They were excised from the DNA of normal cells. As the time between UV-irradiation and pulse-labelling was increased, the frequency of dimer sites associated with the labelled DNA decreased. If the pulse-label was delivered 6 h after irradiation of normal cells or excision-defective xeroderma pigmentosum cells, no dimer sites were detected in the labelled DNA. It has usually been assumed that daughter-strand dimer sites were the result of recombinational exchanges. The assay procedure used in these experiments and in similar experiments of others did not distinguish between labelled DNA containing pyrimidine dimers within the labelled section, and labelled DNA which did not contain pyrimidine dimers but was attached to unlabelled DNA which did contain dimers. The latter structures would arise during normal replication immediately following UV-irradiation of mammalian cells. Calculations are presented which suggest that a significant proportion and conceivably all of the dimer sites associated with the daughter strands may have arisen in this way, rather than from recombinational exchanges as has been generally assumed.     

IS DNA TOPOISOMERASE INVOLVED IN THE UV EXCISION REPAIR PROCESS? NEW EVIDENCE FROM STUDIES WITH DNA INTERCALATING AND NON-INTERCALATING ANTITUMOR AGENTS

Abstract— The effects of selected DNA intercalating and non-intercalating drugs on the UV excision repair process in human fibroblasts have been examined. 9-Amino acridine, acridine orange, quinacrine, doxorubicin (adriamycin), ethidium bromide and actinomycin-D all inhibited the removal of pyrimidine dimers from cellular DNA by inhibiting the incision process as monitored by the nick translation assay and by an endonuclease-sensitive site assay. These agents also partially inhibited incision by the M. luteus endonuclease in an in vitro system. This is the only class of compounds tested to date that appears to block this early step of repair in mammalian cells. The DNA topoisomerase inhibitors, m -amsacrine and VP-16 (etoposide) and the bacterial gyrase inhibitors nalidixic acid and oxolinic acid were shown not to inhibit UV repair. As shown previously, however, novobiocin does block dimer removal and we show here that it is a potent inhibitor of the M. luteus UV endonuclease. While it has recently been demonstrated that many DNA intercalating agents block the strand-passing activity of DNA topoisomerase II giving rise to protein associated DNA strand breaks, the finding that the specific inhibitors of topoisomerase, m -AMSA and VP-16, do not inhibit repair, even though they block this strand passing activity, strongly suggests that inhibition of DNA topoisomerase is not associated with inhibition of DNA repair.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

Rogerio Meneghini

Abstract— Mouse cells (3T3 line) and human fibroblasts are equally sensitive to UV light. At fluences of 2.0–2.5 J/m₂ mouse cells excise only 20% of the pyrimidine dimers as compared to 80% excised by human fibroblasts. This fluence allows 37% survival in both cases. Hence, mouse cells are more resistant to the same burden of unexcised dimers. The reason for this increased tolerance to dimers does not seem to be due to a recombinational mechanism, as judged by the fact that only ca. 5% of the dimers are transferred from parental to daughter strands. The transfer of dimers was measured by the Micrococcus luteus UV endonuclease assay, irradiating cells at Gi to avoid artifacts arising from introduction of dimers in nascent strands. The possibility of other mechanisms being involved in the process of tolerance to DNA lesions is discussed.     

Chlorella virus pyrimidine dimer glycosylase excises ultraviolet radiation- and hydroxyl radical-induced products 4,6-diamino-5-formamidopyrimidine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from DNA

A DNA glycosylase specific for UV radiation-induced pyrimidine dimers has been identified from the Chlorella virus Paramecium Bursaria Chlorella virus-1. This enzyme (Chlorella virus pyrimidine dimer glycosylase [cv-pdg]) exhibits a 41% amino acid identity with endonuclease V from bacteriophage T4 (T4 pyrimidine dimer glycosylase [T4-pdg]), which is also specific for pyrimidine dimers. However, cv-pdg possesses a higher catalytic efficiency and broader substrate specificity than T4-pdg. The latter excises 4,6-diamino-5-formamidopyrimidine (FapyAde), a UV radiation- and hydroxyl radical-induced monomeric product of adenine in DNA. Using gas chromatography-isotope-dilution mass spectrometry and y-irradiated DNA, we show in this work that cv-pdg also displays a catalytic activity for excision of FapyAde and, in addition, it excises 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Kinetic data show that FapyAde is a better substrate for cv-pdg than FapyGua. On the other hand, cv-pdg possesses a greater efficiency for the extension of FapyAde than T4-pdg. These two enzymes exhibit different substrate specificities despite substantial structural similarities.     

COMPARISON OF PHAGE T4 denV ENDONUCLEASE V AND M. Luteus UV-DNA ENDONUCLEASE BY SEROLOGY AND DNA HYBRIDIZATION

Abstract Antibodies were raised in rabbits against purified endonuclease V, the product of the bacteriophage T4 denV gene, which incises DNA at the site of UV-induced pyrimidine dimers. These antibodies cross-reacted with the purified UV-DNA endonuclease from M. luteus in both enzyme-linked immunosorbent assay and Western assays. However, no sequence similarity was detected between the denV gene and M. luteus DNA by hybridization. The two endonucleaes are remarkably similar in enzymatic activity, and their antigenic similarities have been preserved despite differences in their DNA sequences.     

Photorepair of Ultraviolet Radiation (UVR)-lnduced Pyrimidine Dimers in Lens Epithelial DNA of Monodelphis domestica

Abstract— The repair of UV radiation-induced pyrimidine dimers has been measured in lens epithelial DNA of the marsupial Monodelphis domestica using a pyrimidine dimerspecific endonuclease from Micrococcus luteus. Approximately 40% of the initially induced dimers were repaired during 90 min exposures to photoreactivating light. This capacity of the lens epithelium to photorepair pyrimidine dimers may provide a means with which to determine whether pyrimidine dimers in lens epithelial DNA are involved in UV radiation-induced pathologic changes of the lens.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

CHARACTERIZATION OF ANTIBODIES SPECIFIC FOR UV-DAMAGED DNA BY ELISA

Abstract— The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was abrogated. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with O_sO₄. Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin.     

EXCISION OF CYCLOBUTANE DIMERS IN GENOMIC AND EPISOMAL DNA IN HUMAN CELLS

Abstract Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo . Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed cyclobutane dimers were induced at 1.5–2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.     

MORE EFFICIENT EXCISION REPAIR OF PYRIMIDINE DIMERS IN THE SPECIFIC DNA SEQUENCE THAN IN THE GENOME OVERALL IN GOLDFISH CELLS

Excision repair of pyrimidine dimers induced by 254 nm UV was examined in the genome overall and in a specific sequence containing a transfected gene for hygromycin B resistance, in RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. More than 40% of dimers were removed from the specific sequence, while about 20% were removed from the genome overall, within 24 h after exposure to UV (2.5-7.5 J/m2).     

Excision-repair capacity of UV-irradiated strains of Escherichia coli and Streptococcus pneumoniae, estimated by plasmid recovery

Although the biological role of many bacterial repair genes is known, there is still an interest in evaluating the capacity of repair pyrimidine dimers in some strains. For this purpose, we have developed a rapid assay. Cells bearing a plasmid are UV irradiated and incubated to allow recovery. The plasmid DNA is extracted, purified and treated with UV endonuclease from Micrococcus luteus that specifically produces single strand breaks at the site of pyrimidine dimers. The amount of open circular and covalently closed circular forms of the plasmid DNA after treatment and post-incubation provides an estimate of the repair capability of the host strain. The wild type strain and the uvrA mutant of Escherichia coli were used to adjust the assay. The lexA mutant of E. coli has been tested and its repair capability is equivalent to that of wild-type strain. The assay has been extended to Streptococcus pneumoniae, which is naturally deficient in photoreactivation and SOS-like functions. This strain is efficient in the repair of pyrimidine dimers, formed after UV irradiation.     

REMOVAL OF THYMINE-CONTAINING PYRIMIDINE DIMERS FROM UV LIGHT-IRRADIATED DNA BY S1 ENDONUCLEASE

Abstract— S₁ endonuclease was shown to remove thymine-containing pyrimidine dimers from UV-irradiated human DNA, although efficient removal could be demonstated only by using long digestion times, relatively high enzyme concentrations, and irradiation sufficient to yield dimer substitutions in DNA of 1 per 1W300 (dimers/base pair). Neutral and alkaline sucrose gradient analysis of strand break induction by S, of UV-irradiated DNA suggests that recognition of the dimer by S, is the limiting factor in its removal and dimer removal usually results from attack on the dimer containing DNA strand without the induction of a double-strand break.     

SUBSTRATE SPECIFICITY OF A BACTERIAL UV ENDONUCLEASE AND THE OVERLAP WITH IN VITRO PHOTOENZYMATIC REPAIR

Abstract— The action of an endonuclease from Micrococcus luteus , that operates on ultraviolet (UV) radiation damage, overlaps greatly with that of the yeast photoreactivating enzyme: homo and hetero cyclobutyl pyrimidine dimers in DNA are substrate for both enzymes, but pyrimidine adducts or the 'spore photoproduct' in DNA are not.
As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photoreactivating enzyme-dimer complex can prevent nicking by the UV endonuclease. While complex formation between photoreactivating enzyme and dimers in UV-endonuclease-treated DNA is apparently normal, the light-dependent repair step either fails to occur or proceeds at a very low rate. Hence, besides the requirement of a minimum chain length for the function of the photoreactivating enzyme, there is the additional restriction on the position of the dimer in a polynucleotide strand.
Finally, rough approximations of the rate constants, k ₁ and k ₂, for the UV endonuclease indicate that the in vitro UV-endonuclease-dimer complex is relatively unstable, with dissociation of the complex being more probable than hydrolysis of the phosphodiester bond.     

Photoreactivation of cyclobutane dimers and (6-4) photoproducts in the epidermis of the marsupial, Monodelphis domestica

Radioimmunoassays were used to investigate the repair of cyclobutane pyrimidine dimers and pyrimidine (6-4)pyrimidone photoproducts ((6-4] photoproducts) in the epidermis of the South American opossum, Monodelphis domestica. In the absence of photoreactivating light, both types of photodamage were excised with similar kinetics, 50% of the damage remaining 8 h after UV irradiation in vivo. Exposure of UV-irradiated skin to photoreactivating light resulted in removal of most of the cyclobutane dimers and an enhanced rate of (6-4) photoproduct repair. Photoenhanced excision repair of non-dimer damage increases the range of biologically effective lesions removed by in vivo photoreactivation.     

PHOTOREPAIR OF PYRIMIDINE DIMERS IN HUMAN SKIN IN VIVO

Abstract— The exposure of human skin in vivo to UV radiation emitted from a sunlamp induces the formation of pyrimidine dimers. The number of dimers, as detected by UV-endonuclease, decreases following exposure of the UV–irradiated skin to visible wavelengths of light. These results suggest that humans possess a mechanism by which pyrimidine dimers are photorepaired upon illumination of human skin in vivo with visible light.     

INACTIVATION OF NUCLEOTIDE EXCISION REPAIR IN CHINESE HAMSTER OVARY CELLS BY EXPOSURE TO AN ALKYLATING AGENT

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μ M ) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.