Ultraviolet-inactivation of conidia from heterokaryons of Neurospora crassa containing uv-sensitive mutations.

The effect of three UV-sensitive mutations of Neurospora crassa, upr-I, uvs-4 and uvs-6, on the ultraviolet-inactivation of conidia from two-component heterokaryons was investigated. In two-component heterokaryons with wild-type sensitivity to radiation inactivation, all three conidial fractions exhibited similar ultraviolet-inactivation curves. Each UV-sensitive mutation studied uniquely modified the ultraviolet-inactivation curves of conidia from two-component heterokaryons. In heterokaryons heterokaryotic for upr-I, the upr-I mutation was recessive and the repair function determined by the wild type allele was functional to some degree in homokaryotic upr-I conidia. All three conidial fractions of heterokaryons containing upr-I in both components showed increased sensitivity to ultraviolet light. The uvs-4 mutation was recessive and resulted in conidia with increased UV-sensitivity only when included in both components of a heterokaryon. Homokaryotic uvs-4 conidia, which arose from heterokaryons containing both uvs-4 and wild-type components, exhibited wild-type survival. Therefore, as with upr-I, there was a carryover the repair capability to conidia which were genetically UV-sensitive. The uvs-6 mutation, when included in one component of a two-component heterokaryon, resulted in increased UV-sensitivity of both heterokaryotic and homokaryotic uvs-6 conidia. When both components contained uvs-6, the UV-sensitivity of all three conidial fractions was increased and all showed similar inactivation curves. Thus, as with upr-I and uvs-4, there was a carryover of the wild-type repair capability to genetically uvs-6 conidia. Heterokaryon tests for complementation between two non-allelic UV-sensitive mutations showed that in heterokaryotic conidia, complete complementation occurred between upr-I and uvs-4.     

Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA.

UV survival curves of adenovirus 2 using fused, complementing xeroderma pigmentosum (XP) fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusion involved strains in the same complementation group. Extrapolation of this component indicated that at zero dose 3% of the viral plaque-forming units had infected cells capable of normal repair. These results suggest that 3% of the cells were complementing heterokaryons, a value similar to that actually observed by autoradiographic analysis of UV-induced unscheduled DNA synthesis. Thus, heterokaryons formed from XP fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells.     

A human enzyme that liberates uracil from DNA

An enzyme activity which acts specifically on uracil-containing DNA was found in human placenta and cultured fibroblasts. The enzyme liberates uracil from DNA in the presence of EDTA at pH 7.5. Almost equal levels of the activity were found in normal and xeroderma pigmentosum cell lines (complementation group A).     

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of Dna polymerase alpha, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase alpha, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells.     

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.     

Three complementation groups in Cockayne syndrome

After 16 Jm-2 of UV-irradiation non-dividing normal cells recover normal rates of RNA synthesis within 24 h, whereas in cells from donors with Cockayne syndrome (CS) the rate of RNA synthesis gradually declines. Cultures of a mixed population from 2 CS donors were fused with polyethylene glycol; subsequently they were UV-irradiated and RNA synthesis was measured autoradiographically in mono-, bi-, and multinuclear cells. Genetic complementation was indicated by high levels of RNA synthesis in bi- and multinuclear cells when compared with mononuclear cells. Using this assay, 11 CS strains have been assigned to three complementation groups: 2 into group A, 8 into group B and 1 into group C. The strain in group C is derived from an individual who also had xeroderma pigmentosum (XP), and was the sole known representative of XP-complementation group B.     

Thioguanine resistance, ouabain resistance and sister chromatid exchanges in V79/AP4 Chinese hamster cells treated with potassium dichromate

The biological activity of potassium dichromate (K2Cr2O7) was assayed in V79/AP4 Chinese hamster cells by measuring two mutational end points, thioguanine (TG) resistance and ouabain (OUA) resistance, and two non-mutational end points, cytotoxicity and sister chromatid exchanges (SCE). By exposing the cells for 1 h to the chemical, all biological end points examined were affected by the treatment in a dose-dependent manner. Moreover the combined use of the two selective systems indicated that chromium induces base-pair substitutions in mammalian cells in culture.     

Excision repair by human fibroblasts of DNA damaged by r-7, t-8-dihyroxy-t-9,10-oxy-7,8,9,10- tetrahydrobenzo(a)pyrene

Benzo(a)pyrene diol-epoxide I (r-7,t-8,dihydroxy-t-9,10 oxy-7,8,9,10 tetrahydrobenzo(a)pyrene) was used to treat either human adenovirus 5 or cultures of human fibroblasts. The survival of diol-epoxide I treated adenovirus was greater when infecting fibroblasts from normal persons than when infecting fibroblasts from patients with xeroderma pigmentosum (XP). One diol-epoxide I molecule bound per viral genome correlated with one lethal hit as measured using XP fibroblasts.

Normal fibroblasts blocked in semi-conservative DNA synthesis incorporated into their DNA more [³H]thymidine in response to diol-epoxide I treatment than did XP fibroblasts, and also excised more diol-epoxide I from their DNA. All of the effects described above were similar to those obtained when the inactivating agent was ultraviolet light rather than benzo(a)pyrene diol-epoxide I.     

Nuclear deoxyribonuclease activities in normal and xeroderma pigmentosum lymphoblastoid cells

Deoxyribonuclease activities were examined in isoelectric focusing fractions of non-histone chromatin-associated and nucleoplasmic proteins of isolated nuclei of normal human and xeroderma pigmentosum, complementation group A, lymphoblastoid cells using parallel procedures. In the nucleoplasm of both cell lines, a very similar series of both DNA endo- and exo-nuclease activities were found; in chromatin a series of similar endonuclease but no exonuclease activites were present. Several differences were observed in the xeroderma pigmentosum cells, however, notably a striking increase in DNA endonuclease activity in a chromatin fraction at pI 4.6 against linear duplex DNA and a decrease in a chromatin endonuclease activity focusing at pI 7.8.     

A deficiency in the repair of UV and γ-ray damaged DNA in fibroblasts from Cockayne's syndrome

The host-cell reactivation of V antigen production for irradiated adenovirus was examined in fibroblasts from 5 unrelated patients with Cockayne's syndrome (CS) and 2 CS heterozygotes. The fibroblast cultures were infected with either irradiated or non-irradiated adenovirus and subsequently examined for the presence of viral structural antigens using immunofluorescent staining. All CS-homozygous strains showed a reduced host-cell reactivation (HCR) of this viral function for both UV- and γ-irradiated virus. For UV-irradiation of the virus, D₃₇ values expressed as a percentage of that obtained on normal strains, ranged from 14 to 35%. For γ-irradiation of the virus these values ranged from 61 to 80%. These results indicate some defect in the repair of both UV- and γ-ray-induced DNA damage for CS. 1 CS-heterozygote strain tested also showed a reduced HCR for UV-irradiated adenovirus intermediate between that of the patient strain and normal, whereas another CS-heterozygote strain showed an apparently normal HCR level.     

Survival of 60Co-irradiated herpes simplex virus in 15 human diploid fibroblast cell strains

The present study was designed to determine the extent to which herpes simplex virus (HSV) may be utilized to study the repair of DNA damaged by ionizing radiation. We investigated the survival of 60Co-irradiated HSV in cell strains derived from 2 normal controls and 13 patients with a broad range of diseases associated with possible DNA repair deficiencies. Irradiation was performed under two conditions to vary the type of damage incurred by the virus. HSV survival was greatly enhanced when the virus was irradiated in such a way that the indirect effects of ionizing radiation were minimized. We found no correlation between cellular hypersensitivity to ionizing radiation and survival of irradiated HSV. Reduced levels of virus survival were found in only 1 cell strain. When cells were treated with ionizing radiation or UV light prior to infection, no enhancement of virus survival was observed.     

The influence of anoxia or oxygenation on the induction of chromosome aberrations in human lymphocytes by 15-MeV neutrons

Dicentric and total aberration yields induced in human lymphocytes by 15-MeV neutrons under conditions of oxygenation and of anoxia have been fitted to a dose-response curve using the function Y = D + βD². An oxygen-enhancement ratio (OER) ranging from 3.7 at low yields to 1.6 at high yields was calculated from the co-efficients of the dicentric yield curves and evidence is presented which suggests that oxygen does not act as a dose-modifying agent in this system. High dose RBE values of 1.2 and 2.1 with respect to 250 kVp X-rays for oxygenated and anoxic conditions were also obtained. Coefficients for total aberration yield gave similar values to dicentrics for both OER and RBE     

Photodynamic action of chlorpromazine on adenovirus 5: Repairable damage and single strand breaks

Chlorpromazine, a substituted phenothiazine, commonly used as a sedative, has been found to photosensitize the inactivation of human adenovirus 5 to wavelengths of light between 330 and 390 nm. The slope of the inactivation curve is three fold greater when fibroblasts from people having xeroderma pigmentosum (XP) were used as viral hosts than when normal fibroblasts were used, showing that at least two-thirds of the damage produced in the virions is repairable by normal human fibroblasts. The phototreatment of chlorpromazine sensitized virions also results in the production of DNA strand breaks, which correlate fairly well with the production of lethal viral damage as measured in XP fibroblasts. These findings suggest that the photosensitization of the skin observed in patients treated with chlorpromazine might be due to DNA damage.     

Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants at both loci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An anlysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show verly little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104^D1 and mei-41^D5 are located in th eregion 14B13±?14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101^D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103^D1 is between bands 12A1,2 and 12A6,7, and mus(1)-109^A1 is in section 8F3-9A2.     

Effect of dimethylsulfoxide on two synthetic Asn-X-Thr containing substrates of the oligosaccharyltransferase

The enzymatic transfer of oligosaccharides from oligosaccharide lipids to synthetic Asn-X-Thr containing peptides of various length was studied in presence and absence of dimethylsulfoxide. Up to 13.5%, this solvent was found to specifically enhance the efficiency of a ribonuclease heptapeptide to be substrate of the thyroid oligosaccharyltransferase and in contrast, not that of a tripeptide. Circular dichroic analysis performed in various dimethylsulfoxidewater mixtures showed that only the heptapeptide underwent conformational modifications when increasing the concentration in dimethylsulfoxide. Some ordered structure in the immediate vicinity of the Asn-X-Thr signal sequence thus appears of importance in the N-glycosylation process.     

Mutagenic activity of platinum and ruthenium complexes

cis-Dichlorodiammineplatinum(II) [cis-PtCl₂(NH₃)₂] and dichlorotetrakis (dimethylsulfoxide) ruthenium(II) [RuCl₂(DMSO)₄] have been tested as mutagens for strains of Salmonella typhimurium carrying the hisG46 missense mutation. Their activity, which has been compared with the activity of mitomycin C, depends on the presence in the test bacteria of the pKM101 plasmid and is affected in various ways by the function of the excision repair system. More precisely, mitomycin C is mutagenic only for strains with an intact uvr system. cisPtCl₂(NH₃)₂ and RuCl₂(DMSO)₄ are mutagens both for uvrB and uvr⁺ strains, but cis-PtCl₂(NH₃)₂ is more active on the latter, while the converse is true for RuCl₂(DMSO)₄. It seems, therefore, that each drug interacts with DNA by a different mechanism.