Mutation Research/DNA Repair Reports最新文献

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.

A comparative study has been made of the induction and repair of adducts and alkali-labile sites in the DNA of human lymphocytes and granulocytes exposed to the ethylating agents N-ethyl-N-nitrosourea (ENU) and diethyl sulphate (DES).

To evaluate these damages, the human blood cells were treated with highly ³H-labelled ENU and DES, and the resulting ³H-ethyl adducts were analysed via HPLC. Alkali-labile sites introduced in the DNA during treatment with non-radioactive ENU and DES were detected by alkaline elution with fluorometric quantitation of the DNA in the eluted fractions.

All known adducts induced by ENU and DES could be detected by the HPLC methods applied. Furthermore, these adducts were separated from a number of unidentified compounds, because of the improved resolution on the columns used. Most of the adducts were rather persistent during a subsequent incubation period of up to 20 h after treatment, but some partly disappeared (7-ethyladenine and 3-ethyladenine).

The induction of alkali-labile sites in lymphocytes and granulocytes was very similar, but the kinetics of the removal of these sites appeared to be quite different. In granulocytes there was hardly any repair, whereas in lymphocytes, particularly after ENU treatment, a substantial and relatively fast repair was observed. Induction of alkali-labile sites in human lymphocytes and granulocytes occurred also at 0°C; the data suggest that this kind of damage is not a result of enzymic repair processes.

A comparison of the induction and the repair of alkali-labile sites in lymphocytes and granulocytes with those of the various ethyl adducts did not give a clue as to the identity of the adduct that could be responsible for the lability towards alkali.

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.

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 on 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.

Unscheduled DNA synthesis (UDS) was measured autoradiographically in a primary culture of extrahepatic bile duct epithelial cells of Holstein cows following exposure to chemicals known to be capable of developing bile duct cancers in experimental animals, i.e., N-methyl-N′-nitro-N-nitrosoguanidine (MNNG, CAS No. 70-25-7), N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG, CAS No. 4255-77-6), 20-methylcholanthrene (MCA, CAS No. 56-49-5), N-nitrosodimethylamine (DMN, CAS No. 62-75-9) and aflatoxin B₁ (AFB₁, CAS No. 1162-65-8). MNNG and ENNG induced UDS without addition of S9 mixture. MCA elicited UDS only if S9 mixture was added. Regardless of the presence or absence of S9 mixture, DMN failed to induce UDS. DNA repair by AFB₁ was enhanced by the presence of S9 mixture. Therefore, for MNNG, ENNG and high doses of AFB₁ activation by the liver is not necessary to exert genotoxic effects and they seem to be capable of direct action on bile duct epithelial cells in the presence of a bilioenteric fistula or anastomosis.

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.

The incision-rejoining kinetics indicates that 3AB induces a delay in the strand-break rejoining process. In the absence of 3AB the breaks have a lifetime of 1–2 min and this is increased by a factor of 5 in the presence of 5 mM 3AB.

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.

The induction and photorepair of pyrimidine dimers in DNA have been measured in the ultraviolet-irradiated, corneal epithelium of the marsupial, Monodelphis domestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that FS-40 sunlamps (280–400 nm) induced 7.2 ± 1.0 × 10⁻⁵ pyrimidine dimers per kilobase (kb) of DNA per J/m². Following 100 J/m², 50% and > 90% of the dimers were photorepaired during a 10- and 30-min exposure to photoreactivating light (320–400 nm), respectively. In addition ∼ 70% and ∼ 60% of the dimers induced by 300 and 500 J/m², respectively, were repaired by a 60-min exposure to photoreactivating light. The capacity of the corneal epithelium of M. domestica to photorepair pyrimidine dimers identifies this animal as a potentially useful model withwhich to determine whether pyrimidine dimers are involved in pathological changes of the irradiated eye.

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.