Five UV-sensitive mutants obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment of the Staphyloccocus epidermidis W5 strain were characterized phenotypically by assaying their UV- and MNNG-sensitivities, lysogenic inducibility, host-cell reactivation and Weigle reactivation capacities. The results were compared with those of well-characterized Escherichia coli strains, permitting the identification of: 2 mutants that behave as Uvr− Umu−; 1 mutant that appears analogous to Uvr−; 1 mutant that resembles LexA− and 1 mutant that exhibits a RecA− phenotype. The study of these mutants can contribute to the understanding of the repair mechanisms in S. epidermidis.
A temperature-sensitive uvrD mutant, HD323 uvrD4, was isolated from the uvrD mutant HD4 uvrD3. The temperature sensitivity of the uvrD4 gene product was reversible. The suppressor mutation uvrD44 which rendered the uvrD3 mutant temperature-sensitive could be separated from the uvrD3 mutation by replacing the PstI fragment, which encodes the C-terminal half of the UvrD protein. The uvrD44 mutation was found to make host bacteria lethal at non-permissive temperatures only when cloned on a low copy vector pMF3. The nucleotide sequence of the uvrD3 and uvrD4 mutant genes was determined. The nucleotide change found in the uvrD3 at +1235, GAA to AAA, only alters the amino acid sequence from Glu at 387 to Lys. The uvrD44 has another nucleotide change at +1859, GAA to AAA (Glu at 595 to Lys), which is considered to be the suppressor mutation uvrD44.
The expression of the DNA base-excision-repair enzyme uracil-DNA glycosylase in the human hematopoietic system followed a tightly regulated pattern: high enzyme activities were recorded in proliferating bone marrow progenitor cells and in peripheral blood T- and B-cells, both groups of cells requiring the integrity of their genetic information for their proper function. The blood quiescent immunocompetent cells retained their DNA-uracil exclusion capacity, even in the oldest age groups. Peripheral blood mature end cells, granulocytes, platelets and red cells had little activity, consistent with the fact that these cells are anuclear or short-lived, so that no template-primer functions of their DNA are required. Uracil-DNA glycosylase expression is high in all types of human leukemia, providing a selective advantage for survival of leukemic cells. Overall results show that a deficiency of this DNA base-excision-repair pathway is not likely to be an etiopathogenetic factor in the formation of non-random or other chromosomal abnormalities or in the leukemogenesis itself.
Quiescent and concanavalin A-stimulated bovine lymphocytes were subjected to a buoyant density analysis used in excision repair studies. Despite neutral and alkaline rebands to remove replicative contamination, the CsCl gradient profiles of DNA isolated from unstimulated lymphocytes given a 6-h labelling period revealed a small amount of radioactivity in the normal-density region which is indicative of an excision repair process. It amounted to the incorporation of 8,000-20,000 molecules of thymidine per lymphocyte. In a 12-h labelling period the extent of repair incorporation was twice that measured in a 6-h period. The extent of this repair incorporation was not altered significantly during the initial 6 or 12 h of lectin stimulation when DNA-strand breaks normally present in the unstimulated cells are repaired. The same amount of repair activity was found whether the measurements were made on the same day that the lymphocytes were isolated or on the next day following an overnight incubation of the cells in culture medium. These observations indicate that lymphocytes display a spontaneous excision repair activity that proceeds continuously and at a constant rate.
The capacity of a variety of human fibroblasts to incise DNA following exposure to far ultraviolet-light is determined from the rate of single-strand DNA break accumulation in the presence of DNA synthesis inhibitors. We have quantitated incision, one of the early steps in the UV excision repair pathway, in cells form normal, xeroderma pigmentosum groups C, D, G, H and variant individuals, and in the parents of one XPA patient. On the basis of the estimated initial rates of incision the different XP cells examined in this work can be marked as follows: XP variant ⋙XPH > XPD > XPC > XPG > XPA. In each cell strain breaks accumulate immediately after irradiation over a range of 0.5–20 Jm−2 with the exception of the XPC strain examined, where there is an initial delay of 15 min. The rate of incision in XPA heterozygote cells is roughly half that of normal fibroblasts. Analysis of the kinetics of break accumulation over short intervals after irradiation permits estimation of the apparent enzymatic parameters, Km and Vmax, for the incision step. The approximate values of Km and Vmax for normal and XP variant are similar while for the heterozygotes of an XPA individual Km values are normal (around1 Jm−2), but there is only half the amount of normal enzyme activity. XPD and H cells express low levels of active enzyme, between 5 and 15% of that of the normal, but while the Km of KPH is very similar to that of normal cells, that of two XPD strains examined is between 2- and 3-fold higher.
A dodecadeoxynucleotide of defined sequence containing O4-methylthymine was labeled at the 5' end with [32P] by the reaction with (gamma-32P]ATP and polynucleotide kinase. Extracts prepared from bacterial and mammalian sources such as the human cell lines, HeLa and HT29, and rat liver were incubated with the labeled, methylated dodecamer to determine the extent of repair of the lesion. The labeled, demethylated dodecamer was separated from the labeled methylated dodecamer on a reverse-phase column using a shallow methanol gradient. There was complete repair of O4-methylthymine by the E. coli alkyltransferase upon incubation for 4 h at 37 degrees C. There was no detectable amount of demethylated product formed upon incubation with HeLa or HT29 cell extract for the same incubation period. There was also no repair of the O4-methylthymine lesion in the presence of crude rat-liver extract. However, the rat-liver extract alone degraded the methylated substrate completely, and the assay had to be conducted in the presence of NaF, AMP and unlabeled, nonmethylated dodecamer to prevent this. The results obtained from this assay, which is at least an order of magnitude more sensitive than previous methods, are in agreement with previous results that the mammalian alkyltransferase is specific for O6-alkylguanine repair.
The molecular basis of sensitivity of ionizing radiation and other damaging agents is not clearly defined in eukaryotes. While a large number of mutants have been described only a few have been demonstrated to have a defect in the repair of damage to DNA. An interesting characteristic of a sub-group of these mutants, in different species extending throughout the phylogenetic scale, is the presence of damage-resistant DNA synthesis. This phenomenon is observed in cells from individuals with the genetic disorder ataxia telangiectasia, in HeLa cells treated with fluorodeoxyuridine prior to UV irradiation, in mutants of the fungus Neurospora crassa, the slime mould Dictyostelium discoideum, the fruit fly Drosophila melanogaster and possibly in the "wasted" mouse mutant. In the case of ataxia telangiectasia sensitivity is only observed to ionizing radiation or radiomimetic chemicals whereas sensitivity to a wider spectrum of mutagens is reported for the lower eukaryotic mutants. In all cases a reduced inhibition of DNA synthesis is obtained after exposure to an agent to which the cell type is hypersensitive. It is unclear how damage-resistant DNA synthesis contributes to increased sensitivity in these cells, but is unlikely to be the major mechanism predisposing to radiation-induced cell death. The description of a derivative of an ataxia telangiectasia cell line with normal sensitivity to radiation but still maintaining resistant DNA synthesis partially uncouples radioresistant DNA synthesis and radiosensitivity. This paper is designed to review the phenomenon of damage-resistant DNA synthesis in a number of mutants.
In order to discover whether the nuclear recombinational repair pathway also acts on lesions induced in mitochondrial DNA (mtDNA), the possible role of the RAD50, −51, −52, −55 and −56 genes on the induction of rho− mutants by radiations was studied. Such induction appeared to be independent of this pathway. Nevertheless, an efficient induction of respiration-deficient mutants was observed in γ-irradiated rad52 diploids. We demonstrate that these mutants do not result from a lack of mtDNA repair, but from chromosome losses induced by γ-rays. Such an impairment of the respiratory ability of diploids by chromosome losses was effectively observed in the aneuploid progeny of unirradiated RAD+ cdc6 diploids incubated at the restrictive temperature.
Excision repair kinetics of UV-induced pyrimidine dimers in DNA of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes were compared to unstimulated lymphocytes using a dimer-specific endonuclease from Micrococcus luteus in conjunction with agarose gel electrophoresis. Removal of pyrimidine dimers could be detected within 6 h after irradiation only PHA-stimulated lypmhocytes. However, incorporation of [3H]thymidine as UV-induced unscheduled DNA synthesis was detected in the unstimulated lymphocytes in the 6-h period. The number of pyrimidine dimers remaining in unstimulated lymphocytes was approximately 85% after 24 h as compared to less than 25% in stimulated cells.
A new gene, mus-16, is determined by the nitrogen mustard-sensitive Neurospora mutant of Baker, Parish and Curtis (1984) which is defective in the removal of DNA-DNA and DNA-protein crosslinks. This gene is on the left arm of linkage group V between caf-1 and lys-1. The mus-16(JMB) mutant is sensitive to the alkylating agents methyl methanesulfonate (MMS) [dose reduction factor (drf) 8–10 ×], N-methyl-N′-nitro-N-nitrosoguanidine (drf 5–6 ×), the amino acid histidine and the drug hydroxyurea. It is not sensitive to ultraviolet-light, γ-irradiation, or mitomycin C (MMC). It shows normal spontaneous mutation rates but increased induction of mutation by MMS. Homozygous crosses are barren, showing no signs of sporulation. Mitotic spontaneous chromosome instability is increased. The mus-6 mutation is similar to several non-excision repair-defective mutants in Neurospora. Some of these may be defective in repair of alkylation damage. The MMC data supports earlier data that in fungi MMC is incapable of forming DNA-DNA crosslinks.
Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt+ colonies were selected, and cell lines established. Several lines were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95,000-dalton UvrA-related protein. The expression of uvrA was found to be 50-100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the "mammalian" UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA+ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA+ cell lines were examined for their UV-resistant phenotype and not unexpectedly were found not to be reverted to a state of repair proficiency.
The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, on DNA-repair processes has been investigated after treating V79 hamster cells with methyl methanesulphonate (MMS). Repair activity was observed as changes in DNA-strand break levels. MMS induces transient strand breaks, the level of which slowly decreases with time. Addition of 3AB leads to a rapid increase in the number of breaks. The level of breaks increases linearly with time until it suddenly levels off. Increasing the concentration of 3AB does not change the slope of this curve, but the steady-state level of breaks increases.
Survival and mutagenesis caused by 5-azacytidine was studied in Escherichia coli. Survival was partially lexA- and recA-dependent and was decreased by the presence of a DNA (cytosine-5)methyltransferase. The dcm, MspI, and EcoRII methyltransferase genes all decreased survival. There was no direct relationship between amount of methylase enzyme present and cell survival, but only plasmids containing a methylase gene sensitized cells to 5-azacytidine. Survival was not affected by uvrA, uvrB or umuCD mutations. Induction of sulA::lacZ fusions by 5-azacytidine was inhibited in strains containing elevated levels of DNA methylase. Cells resistant to 5-azacytidine when they contained a plasmid specifying the EcoRII methylase were sensitive if the plasmid specified the complete EcoRII restriction-modification system. The mechanism of cell death in these situations is therefore different. Mutation of the rpoB gene by 5-azacytidine was studied. The mutation rate was decreased by the presence of recA and lexA mutations. Mutation in umuCD had little effect on the mutation rate. The recA430 mutation, which does not support SOS-dependent mutagenesis induced by UV light, does support 5-azacytidine induced mutagenesis. The presence of DNA (cytosine-5)methyltransferase had no effect on the mutation rate caused by 5-azacytidine treatment. The mutagenic and lethal lesions caused by 5-azacytidine in the absence of methylase therefore differ from the lethal lesions that occur in the presence of methylase. The former could be due to the opening of the 5-azacytosine ring in DNA. Cell death in the presence of methylase could be due to tight binding of methylase to azacytosine containing DNA as well as inhibition of induction of the SOS response.
The ionising radiation-sensitive mutants irs 1, irs 2, irs 3, xrs-1 (or xrs-7, EM7 and XR-1 were fused to wild-type cells or to each other in pairs to create hybrid cells. These hybrids were checked chromosomally and their X-ray sensitivity tested. Each mutant was found to be recessive to wild-type and to complement the X-ray sensitivity of the other mutants. Thus there appear to be at least 6 complementation groups for ionising radiation sensitivity in Chinese hamster cells.
The repair of DNA alkylation damage in human cells is poorly understood. We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture. We have also established conditions necessary for short-term culture of human lymphocytes. Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF). The amount of damage induced by a given concentration of methyl methane-sulfonate (MMS) is dependent upon cell number per ml of growth medium. The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single strand breaks and alkali-labile lesions. Repair of this damage after appropriate recovery periods is also detectable. The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment. This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes.
The isolation and characterisation of mutants of Aspergillus nidulans showing resistance to MNNG is described. Such isolates were stable through prolonged subculture in the absence of the selective agent, and resistance segregated as an allele of a single gene in meiotic and mitotic analysis. MNNG-resistant strains showed an increase in resistance to EMS and UV irradiation but no cross-resistance to MMS was detected. Possible mechanisms of resistance to alkylating agents are discussed.
Escherichia coli lost its colony-forming ability when suspended in Tris/NaOH or Tris/Mg2+ buffers of pH 10.0 and 4.0, respectively. A significant decrease in the survival of radiation-sensitive mutants recA, polA, res, rer and lexA was observed as compared to their wild-type counterpart under these conditions. The alkali-injured cells were found to recover when incubated at 37°C for 2 h in 0.05 M phosphate buffer of pH 8.0, whereas no such liquid holding recovery was observed in recA and lexA mutants. Recovery in phosphate buffer was not affected by metabolic inhibitors. As a result of alkali treatment, the sensitivity of bacteria to ultraviolet light (UV) was enhanced. However, on incubation for 2 h in recovery buffer at 37°C, the bacteria regained partial UV resistance. Bacteria exposed to alkaline environment exhibited an enhanced level of mutagenesis. Contrary to the treated wild-type, the mutants recA and lexA did not exhibit any increase in the mutation frequency. Alkali treatment to GC → AT transition mutants of Salmonella typhimurium, TA102 and TA104 resulted in the highest number of revertants per plate.
An SV40-transformed Fanconi's anaemia (FA) cell line, GM6914, exhibits approximately 2.4-fold increased sensitivity to the cytotoxic effects of nitrogen mustard (NM) when compared with the normal line, MRC5-V1. Host cell reactivation of NM-treated plasmid has been investigated using transient expression vectors which contain the chloramphenicol acetyltransferase (CAT) gene. In both cell types there is a similar, dose-dependent reduction in CAT expression which correlates with an increase in NM-induced DNA-interstrand crosslinking. The data are consistent with two possible mechanisms for inactivation of the plasmid. Either a single crosslink anywhere within the plasmid is sufficient to prevent transcription of the cat gene. Alternatively, inactivation may result from some other more prevalent NM-induced lesions within the cat coding sequence.
Various cell types in spermatogenesis exhibit differential sensitivity to radiation-induced DNA damage. The investigation of DNA radiosensitivity in vitro is complicated by the heterogeneous population of male germ cells (MGC) present in isolated single-cell suspensions. In the present investigation, the neutral elution technique was used to assess gamma-irradiation-induced DNA double-strand damage (DSD) in spermatogonia and preleptotene spermatocytes (SG/PL), pachytene spermatocytes and spermatid spermatocytes, as well as in MGC. In addition, the capability of these cell types to repair DNA double-strand damage was investigated. Based on the well established timing of the rat spermatogenic cycle, the DNA of specific cell populations was labeled using tritiated thymidine. DNA from labeled cells was determined isotopically, whereas total DNA was quantitated using a fluorometric method. DSD was induced in a dose-dependent manner in the heterogeneous population as well as in the labeled cell populations. SG/PL were more sensitive to gamma-irradiation-induced DSD than either the heterogeneous MGC population, pachytene or spermatid spermatocytes. Each cell type exhibited a similar capability to repair DSD following exposure to 3000 rad; repair was rapid (maximal within 45 min) and incomplete (less than 40%). Only pachytene spermatocytes exhibited significant repair following exposure to 6000 rad. Since a difference in sensitivity to radiation-induced DSD was demonstrated, the capability of each cell type to repair a similar initial frequency of strand damage was investigated. SG/PL, pachytene and spermatid spermatocytes differed in their capability to repair similar levels of strand damage. However, the difference in dose required to achieve equal damage may have contributed to other cellular effects, thus altering repair. In summary, a model is described that permits the evaluation of genotoxic responses in specific populations of spermatogenic cells within a heterogeneous cell suspension. The ability of specific cell types to repair gamma-irradiation-induced DNA double-strand damage is demonstrated.
DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TGr) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (ouar) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more ouar mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.
