Mutation Research/DNA Repair Reports最新文献

Autoradiographic analysis of unscheduled DNA synthesis in response to DNA damage produced by relatively high doses of the direct-acting, monofunctional alkylating agent, MMS, (4.5 mM) and UV (40 J/m²) demonstrates that established cell lines, derived from the mei-9^a and mus201^D1 excision repair-deficient strains of Drosophila melanogaster, perform no measurable incorporation of [³H]thymidine into repair patches, in accordance with the observations made for the corresponding primary embryonic cells derived from the two mutagen-sensitive strains of flies. These established cell lines can therefore be used as appropriate models for both examination of the biochemical basis of the genetic defects in the mei-9 and mus201 mutations and the role of excision-repair processes in spontaneous and induced mutation induction in eukaryotic cells.

The plasmid pBR322 was treated with BamHI, PvuII and γ-irradiation to generate double-strand breaks (dsb) containing differently structure ends. Transformation efficiencies, mutation frequencies and clone analyses of enzymatically damaged DNA are compared with the corresponding results from radiolytically damaged DNA. In E. coli K-12 SFX, the yield of transformants produced by the action of BamHI, PvuII and γ-irradiation (30 Gy) is 4.3%, 0.14%, and 0.10%, respectively. The survival of open circular DNA (ocDNA) produced by 30 Gy is 1.3%. The transformation efficiencies show only a slight dependence on SOS induction and on the RecA protein. Mutation frequencies to tetracycline sensitivity (tet^S) per surviving plasmid are 2.6% (BamHI), (PvuII) and 0.2% (γ-irradiated DNA with 30 Gy containing ∼ 50% oc DNA and 50% linearized (lin) DNA). The mutation frequency is low at all radiation doses studied (1–50 Gy). Only 15% of the DNA of the tet^S mutants from γ-irradiated plasmids contained deletions whereas with enzymatically damaged DNA, 30–50% (BamHI) or 90% (PvuII) contained deletions. In all cases, all deletions comprised 500–1700 base pairs (bp). After SOS induction of the host cells, the mutation frequency of γ-irradiated plasmids increased by a factor of 4, whereas that of the enzymatically damaged plasmids did not change.

For the repair of the enzymatically linearized DNA 2 recombinational pathways are discussed which lead to deletant (pathway I) and non-deletant transformants (pathway II). In addition, BamHI-linearized plasmids may be repaired by enzyme-induced or spontaneous circular alignment followed by ligation. The high percentage of deletions of the tet^S mutations for PvuII-linearized DNA with the blunt ends is explained by the illegitimate or site-specific recombination pathway I (see text). The lower percentage of deletions of the tet^S mutations with BamHI-linearized DNA with short cohesive ends (4 bp) is proposed to be due to a greater contribution of pathway II and/or by circular alignment followed by ligation. The very small yield and the low percentage of deletant mutations of tet^S mutants from radiolytically damaged DNA is proposed to be due to the large overlapping ends (16–100 bp) of the linDNA which easily leads to circular alignment followed by excision repair. The repair of radiolytically produced ocDNA is predominantly due to excision repair. In agrement with this interpretation is the observation that SOS induction of the host increases the mutation incidence of radiolytically damaged DNA but not of enzymatically damaged DNA.

The mutagenicity of 1,2-dibromoethane (EDB) to Escherichia coli was reduced by the UV light-induced excision repair system but unaffected by the loss of a major apurinic/apyrimidinic site repair function. At high doses, 70–90% of the EDB-induced mutations were independent of SOS-mutagenic processing and approximately 50% were independent of glutathione conjugation. The SOS-independent mutations induced by EDB were unaffected by the enzymes that repair alkylation-induced DNA lesions. EDB-induced base substitutions were dominated by GC to AT and AT to GC transitions. These results suggest that EDB-induced premutagenic lesions have some, but not all, of the characteristics of simple alkyl lesions.

The similarity of the adaptive response and the methyltransferase component in bacterial strains from different phylogenic groups was investigated. An adaptive response with induction of transferase activity was found for the first time in the soil bacteria P. aeroginosa and X. maltophilia. Polyclonal antibodies against the E. coli ada protein were used to investigate the structural similarity of the transferases from several bacterial strains with adaptive responses and inducible transferase acitivity. These antibodies cross-reacted with transferase from M. leteus and P. aeroginosa but not with proteins from other related bacteriam, and not with human transferase. The phylogenic relationships of bacteria with adaptive responses suggest that the response likely was present in the common ancestor of eubacteria. The restricted antibody cross-reactivity may reflect the dual role of the E. coli ada protein not only in DNA repair but in positive gene regulation.

The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radiosensitivity from radioresistant DNA synthesis.

Various cell types in spermatogenesis exhibit differential sensitivity to radiation-induce DNA damage. The investigation of DNA radiosensitivity in vitro is complicated by the heterogneous population of male germ cells (MGC) present in isolated single-cell suspensions. In the present investigation, the neutral elution technique was used to assess γ-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 metod. 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 γ-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 rapdi (maximal within 45 min) and incomplete (< 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 γ-irradiation-induced DNA double-strand damage is demonstrated.

A radioimmunoassay was used to study the induction and repair of damage produced by the photolysis of (6-4) photoproducts in normal and UV-sensitive human cells: Photochemical conditions were established to optimize the production of photolyzed (6-4) photoproducts in human cell DNA with minimal induction of other photoproducts. The repair of this photoproduct, presumed to be a Dewar pyrimidinone, was similar to that determined for the (6-4) photoproducts, with most of the antibody-binding sites removed within 4 h post-photolysis. Whereas xeroderma pigmentosum group A cells were deficient in the repair of this lesion, an XP variant and two cell lines selectively hypersentitive to UVB-irradiation were shown to have normal repair. The radioimmunoassay was further used to demonstrate the alkali-lability of the (6-4) photolysis product.

A V79 Chinese hamster cell line XR-V15B exhibiting hypersensitivity to X-ray has been isolated and characterized. Additionally to increased X-ray-sensitivity (approximately 8-fold, as judged by D₁₀ values), cross-sensitivity to bleomycin (3-fold increase), 4NQO (3-fold), H₂O₂, EMS, MMS (2-fold) were observed also. No increased sensitivity to UV and MMC was found. Genetic complementation analysis indicates that XR-V15B belongs to the same complementation group as the X-ray-sensitive (xrs) mutants of Chinese hamster ovary (CHO) cells described by Jeggo (1985). Biochemical analysis of XR-V15B confirms this finding: the mutant showed a decreased ability to rejoin double-strand breaks induced by X-ray as measured by neutral elution. After 4 h of repair more than 50% of the double-strand breaks remain in comparison to 3% in V79 cells. No difference was observed wild-type and XR-V15B cells in the initial number of single-strand breaks induced, in the kinetics of their rejoining and in the final level of unrejoined single-strand breaks. Treatment with 5-azacytidine did not have an effect on the reversion frequency of XR-V15B, contrary to the results obtained with the xrs mutants. XR-V15B has been grown in continuous culture for more than 3 months without evidence of reversion. The mutation induction by X-ray irradiation at the HPRT locus is not significantly increased in the mutant, but at doses giving the same degree of cell killing, XR-V15B cells are hypomutable.

We obtained a monoclonal antibody directed against UV-induced DNA damage. Analysis of the antigenic determinant in UV-irradiated DNA recognized by this antibody, 64M-1, revealed that it bound UV-irradiated oligo- or poly-nucleotides containing thymine—thymine or thymine—cytosine sequences. The antibody failed to bind DNA irradiated with 313 nm UV in the presence of acetophenone, which contained predominantly thymine dimers as DNA damage. The binding activity of this antibody to 254-nm UV-irradiated DNA decreased with 313-nm UV irradiation, and the decrease of this binding activity correlated with the decrease of fluorescence corresponding to (6-4) photoproducts. These results suggest that the antigenic determinant recognized by this monoclonal antibody is a (6-4) photoproduct. Using autoradiography with ³H-antibody, we could detect the formation of the (6-4) photoproduct in individual human cells irradiated with 254-nm UV doses as low as 20 J/m².