Excision Of Dimers Research Articles

Cyclobutane-pyrimidine dimer excision in UV-sensitive CHO mutants and the effect of the human ERCC2 repair gene

Using a radiochromatographic assay, we have examined cis-syn cyclobutane-pyrimidine dimer removal after ultraviolet irradiation in cell lines representative of the first 6 complementation groups of Chinese hamster ovary DNA nucleotide excision repair mutants. AA8, the CHO cell line from which these mutants were derived, consistently showed normal dimer excision for a rodent cell. The mutants uniformly exhibited no significant dimer excision within the limits of determination. Additionally, V-H1, a mutant belonging to complementation group 2 and derived from V79 hamster cells, exhibited no dimer excision. Two UV5 derived transformants that carry the complementing human ERCC2 repair gene showed a capacity for dimer excision comparable to the AA8 wild-type cells.

DNA excision-repair ability of embryonic fibroblasts measured by UDS and thymine dimer excorporation in four inbred mice strains

Differences in the excision-repair capability of embryonic fibroblasts of four inbred strains of mice following various degrees of UV irradiation were assessed. Two methods of determination were used: (1) the incorporation of 3H-thymidine during unscheduled DNA synthesis (UDS) as measured by an autoradiographic technique; (2) the rate of excision of thymine dimers ( TT ) in the acid-insoluble fraction of the cellular DNA as determined by a dimer-specific radioimmuno-assay. Based on UDS, the repair rates of the four strains could be ranked in decreasing order as follows: DBA/2 (DB); C57BL/ 6J (B6); AKR/N (AK); CBA/J (CB). The calculated rate for DBA/2 (DB) is approximately twice that of CBA/J (CB). The determination of the TT excision rate indicates that 72 h after irradiation a maximum of 50% of the original UV-induced dimers in the DB strain could be repaired. In the three remaining strains the relatively reduced repair rates of 15% – 40% did not differ significantly.

Inhibition by metals of X-ray and ultraviolet-induced DNA repair in human cells

A number of metals have been shown to be involved in the etiology of animal and human neoplasms. The molecular mechanisms have not yet been determined, but the observed plethora of genetic effects observed following treatment of mammalian cells with metals clearly indicates the possibility that metals can exert their effects at least partially at the level of DNA metabolism. Several studies have suggested that metal treatment may inhibit normal DNA repair processes in procaryotic and eucaryotic cells but a systematic study of this question has not previously been conducted. The present study surveyed the ability of 15 metal salts to interfere with repair of X-ray or UV-induced DNA damage in HeLa cells. Hg+(+), As++(+), Cu+(+), Ni+(+), Co+(+), and Cd+(+) were shown to inhibit the excision of pyrimidine dimers from DNA and to do so in a dose-dependent fashion. Inhibition of repair by only Ni+(+) and Co+(+) resulted in the accumulation of long-lived DNA strand breaks suggestive of a block in the gap-filling stage of repair. Ability to inhibit repair was not correlated with cytotoxicity. X-ray repair was sensitive to Hg+(+), Ni+(+), As++(+), Ga+(+), Zn+(+), and Mo(VI). All inhibitory metals inhibited closure of single strand DNA breaks. Ga+(+) appeared, in addition, to inhibit a later step involving chromatin reconstitution. These findings support the notion that interference of DNA repair processes may be a consequence of exposure of mammalian cells to certain metals. This may be a factor in the etiology of metal-associated carcinogenesis.

In UV-irradiated Escherichia coli PQ35 overproducing the RecA protein, expression of the sfiA gene and dimer excision are alleviated.

Escherichia coli PQ35 cells carrying the sfiA-::lacZ operon fusion were transformed either with a multicopy plasmid containing the recA gene (pHSG262 recA) or with a multicopy plasmid alone (pHSG262). Both transformants were UV irradiated. Then induction of the sfiA gene and dimer excision were followed. Amplification of the recA gene partly inhibited both sfiA gene induction and dimer excision. The following interpretation of this phenomenon is proposed. When the RecA protein is in abundance, pyrimidine dimers are quickly masked by it. The masked dimers are less efficiently distinguished by excision nuclease and do not provide the induction signal. Due to this, induction of the sfiA gene as well as dimer excision are inhibited early.

Repression of damage-inducible ( din) genes by the lex A3 mutation or by plasmid carrying the lex A gene; effect on pyrimidine dimer excision in UV-irradiated Escherichia coli

Dimer excision was followed in Escherichia coli K-12 AB1157 DM49 lexA 3 mutant (whose represser is not cleavable with RecA protease), and in E. coli K-12 AB2497[pGC3] carrying the cloned lexA gene. In either case din genes could not be efficiently derepressed. In such cells ultraviolet (UV) irradiation caused an extensive DNA degradation, which was not observed in cells with derepressed din genes. Even after a high UV dose (70 J/m 2) dimers were being excised efficiently. However, progressive DNA degradation interfered with the precise detection of unexcised dimers. We conclude that induction of din genes is required for filling some of the gaps and for prevention of DNA degradation, but not for excision itself.

Excision repair characteristics of denV-transformed xeroderma pigmentosum cells

Introduction of the denV gene if 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 were 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-endonucleasse 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 to be accompanied by the resynthesis of a region ∼ 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.

DNA damage and repair in normal, xeroderma pigmentosum and XP revertant cells analyzed by gel electrophoresis: excision of cyclobutane dimers from the whole genome is not necessary for cell survival.

Human fibroblasts were embedded in microscopic-sized agarose beads and lysed in situ to produce chromosome-sized DNA trapped inside the beads. Conventional alkaline agarose gel electrophoresis of trapped DNA from cells damaged by X-rays before embedding gave mol. wts similar to those obtained with alkaline sucrose gradients. When cells were irradiated with UV light before embedding in agarose and incubated with UV endonuclease to cleave cyclobutane pyrimidine dimer sites, UV damage was detectable by gel electrophoresis over a range of 2.6-52 J/m2 as a linear function of dose, and repair was detectable within 6 h of irradiation. Two independently derived UV-resistant xeroderma pigmentosum (XP) revertants did not remove cyclobutane dimers up to 48 h after irradiation. Incubation of normal and XP revertant cells with cytosine arabinoside after UV irradiation resulted in similar numbers of single-strand breaks; these breaks represent repair of non-dimer photoproducts. Evidently, excision of cyclobutane pyrimidine dimers from the whole genome is not necessary for survival of human cells after UV irradiation; rather, repair of non-dimer photoproducts such as photoproducts in the genome as a whole or cyclobutane dimers in a small number of genes may be more biologically important.

The effect of temperature and wavelength on production and photolysis of a UV-induced photosensitive DNA lesion which is not repaired in xeroderma pigmentosum variant cells.

Abstract— Ultraviolet light causes a type of damage to the DNA of human cells that results in a DNA strand break upon subsequent irradiation with wavelengths around 300 nm. This DNA damage disappears from normal human fibroblasts within 5 h, but not from pyrimidine dimer excision repair deficient xeroderma pigmentosum group A cells or from excision proficient xeroderma pigmentosum variant cells. The apparent lack of repair of the ultraviolet light DNA damage described here may contribute to the cancer prone nature of xeroderma pigmentosum variant individuals. These experiments show that the same amount of damage was produced at 0°C and 37°C indicating a photodynamic effect and not an enzymatic reaction. The disappearance of the photosensitive lesions from the DNA is probably enzymatic since none of the damage was removed at 0°C. Both the formation of the lesion and its photolysis by near ultraviolet light were wavelength dependent. An action spectrum for the formation of photosensitive lesions was similar to that for the formation of pyrimidine dimers and(6–4) photoproducts and included wavelengths found in sunlight. The DNA containing the lesions was sensitive to wavelengths from 304 to 340 nm with a maximum at 313 to 317 nm. This wavelength dependence of photolysis is similar to the absorption and photolysis spectra of the pyrimidine(6–4) photoproducts

Chromosome and DNA damage and their repair in higher plants irradiated with short-wave ultraviolet light

In this review the authors present only their own results. They include the determination of the duration of the different stages of the cell cylce in UV-irradiated barley cells, the effect of different UV doses on the frequency of chromosome aberrations in barley, the increase in UV-induced chromosome aberration frequency induced in barley by caffeine and the effect of UV doses on the induction of pyrimidine dimers and sites sensitive to UV-endonuclease action (ESS) in barley cells and Nicotina tabacum protoplasts. In addition, the excision of pyrimidine dimers and ESS after irradiation with various doses of UV, unscheduled DNA synthesis in N. tabacum protoplasts and the correlation between the induction of pyrimidine dimers in DNA and the frequency of chromosome aberrations are reported. Data demonstrating that photoreactivation decrease the number of DNA lesions and chromosome aberrations induced by UV are also presented.

The fate of thymine-containing dimers in ultraviolet-irradiated Halobacterium cutirubrum

Extremely halophilic bacteria do not recover in the dark from the effects of ultraviolet radiation, although they can be completely photoreactivated, thus proving that pyrimidine dimers are the cytotoxic photoproducts. We have now shown, by studying the fate of thymine-containing dimers during dark liquid-holding of Halobacterium cutirubrum, that the lack of dark repair in these bacteria is due to their inability to excise such lesions, in contrast to Escherichia coli B. Nevertheless, extensive nonspecific degradation of DNA occurs in H. cutirubrum following ultraviolet irradiation, so the lack of dimer excision is not due to a generalised inability to degrade DNA after such treatment. These findings raise interesting questions concerning the basis of the resistance of halophiles to ultraviolet radiation.

DNA‐PROTEIN CROSSLINKING IN NORMAL HUMAN SKIN FIBROBLASTS EXPOSED TO SOLAR ULTRAVIOLET WAVELENGTHS

Three normal human skin fibroblast cell lines were exposed to the simulated solar UV radiation produced by a fluorescent sunlamp under conditions in which the wavelength components shorter than either 295, 305 or 315 nm were excluded. The level of DNA-protein crosslinks (DPC) was then measured in those cells using the alkaline elution technique either immediately after irradiation or following a 24 h incubation. In each case, cells were exposed to fluences that induce similar levels of DPC. For cells exposed to 10 kJ m(-2) of sunlamp UV > 295 nm, the level of DPC exhibited a 2-5-fold increase following incubation. In contrast, 40-100% of the DPC were removed upon incubation of cells irradiated with either 100 kJ m(-2) of sunlamp UV > 305 nm or 150 kJ m(-2) of sunlamp UV > 315 nm. A major difference between the effects induced by these wavelength regions is that, in addition to DPC, a very high level of pyrimidine dimers is also produced by sunlamp UV > 295 nm, whereas much lower dimer yields result from treatment with either sunlamp UV > 305 nm or sunlamp UV > 315 nm. A potential role for type II DNA topoisomerase in the formation of these DPC resulting from either the change in conformational structure caused by the presence of a high level of dimers or an involvement of this enzyme in dimer excision repair is discussed.

Inhibition of pyrimidine dimer excision in ultraviolet-irradiated Escherichia coli overproducing RecA protein

Escherichia coli Br Hcr + cells transformed with the recombinant multicopy plasmid pBR322 carrying recA gene contain increased amounts of RecA protein. When these cells were UV-irradiated, excision of pyrimidine dimers was reduced by about 50%. It is suggested that the damaged DNA strands may be coated with RecA protein which makes them insensitive to the action of the uvrABC excision nuclease.

General characteristics, molecular and genetic analysis of two new UV-sensitive mutants of Chlamydomonas reinhardtii

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.

DNA synthesis in irradiated mammalian cells.

One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v.-induced inhibition of replicon initiation.

Pyrimidine dimers in Drosophila chromatin become increasingly accessible after irradiation

A prokaryotic DNA-repair enzyme has been utilized as a probe for changes in the accessibility of pyrimidine dimers in Drosophila chromatin following UV irradiation. The results demonstrate a rapid cellular response to physiologically relevant doses of radiation which results in at least a 40% increase in accessible dimers. This increase occurs in two incision-deficient mutants which indicates that the excision-repair process, at or beyond the incision step, is not required or responsible for the increase. In the absence of excision the increase in accessibility persists for a least 2 days following irradiation. The observed increase in accessibility is inhibited by both novobiocin and coumermycin. These inhibitors do not inhibit the initial rate of incision, but do reduce dimer excision measured over more extended periods. A pre-incision process is proposed which actively exposes DNA lesions to excision repair. A fraction of the genome is postulated to be accessible without the intervention of that process.

Immunochemical determination of an initial step in thymine dimer excision repair in xeroderma pigmentosum variant fibroblasts and biopsy material from the normal population and patients with basal cell carcinoma and melanoma.

A monoclonal antibody specific for u.v.-induced thymine-thymine dimers in single-stranded DNA has been used in an enzyme immunoassay to investigate the loss of antigenicity associated with repair of this lesion in the first 2 h following 10 J/m2 254 nm radiation. Variances of +/- 10% for the method and +/- 6.5% for individuals were established using primary cultures of biopsies from healthy individuals. No differences in the rate of loss of antigenicity was observed between 20 normal lymphocyte samples and 10 normal skin biopsies. Of three xeroderma pigmentosum (XP) variant cell lines tested, GM3617 could not be distinguished from normal cells but GM1227 and GM3053 showed lower rates of loss than any of the healthy samples. When the group mean values were compared there was no significant difference between normals and biopsies from sun-shielded skin areas from 16 basal cell carcinomas but similar material from 10 melanoma patients showed a significantly reduced (P = 0.001) rate of loss of antigenicity. Since the rate of loss of antigenicity in normal and XP variant cells reflected their relative abilities to perform unscheduled DNA synthesis, our results suggest that some melanoma patients may also have a minor deficiency in an early stage of excision repair.

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 (As 2O 3) and pentavalent sodium arsenate (Na 2HAsO 4) in normal human and xeroderma pigmentosum (XP) fibroblasts. The presence of As 2O 3 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 μg/ml and 100% at a supralethal dose of 5 μg/ml. Correspondingly, As 2O 3 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. Na 2HAsO 4 (As 5+) was approximately an order of magnitude less effective in preventing all the above repair events than As 2O 3 (As 3+) 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 As 2O 3 (0.5 μg/ml) and Na 2HAsO 4 (5 μg/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 As 2O 3 and Na 2HAsO 4 killing by a yet unidentified mechanism.

Pyrimidine dimer excision repair of DNA in Bacteroides fragilis wild-type and mitomycin C-sensitive/UV-sensitive mutants.

An enzyme preparation purified from Micrococcus luteus was shown to be specific for UV-induced pyrimidine dimers and was suitable for the detection of DNA excision repair systems. The wild-type Bacteroides fragilis Bf-2 strain and a mitomycin C-sensitive mutant (MTC25) had constitutive dimer excision systems which functioned efficiently under anaerobic and aerobic conditions. A UV-sensitive mutant (UVS9) had markedly reduced levels of the constitutive dimer excision systems under anaerobic and aerobic conditions. Since liquid holding recovery under aerobic conditions was inhibited by chloramphenicol whereas the final level of excision repair in B. fragilis Bf-2 was not affected, it is concluded that pyrimidine dimer removal is not the process responsible for increased physiological aerobic liquid holding recovery.

Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts.

To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Excision of pyrimidine dimers from the DNA of Neurospora.

Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmunoassay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers. UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency.