Repair of DNA alkylation adducts in mammalian cells

Carcinogenic alkylating agents, including nitrosamines, are able to alkylate DNA at various sites. This review presents evidence of the high degree of specificity in the type of DNA damage induced by various N-nitroso compounds and in the DNA repair processes among tissues or cells of different species. The O6-alkylguanine DNA alkyltransferase activity in various human and rodent tissues is discussed as well as the detection of O6-methylguanine in human DNA, using monoclonal antibodies and radioimmunoassay. The relevance of these findings to the mechanisms of cancer induction by nitrosamines is discussed.     

DNA damage and repair in mammalian cells exposed to p-hydroxyphenylpyruvic acid

Tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine metabolism in which the fumarylacetoacetate hydrolase enzyme is defective. This disease is clinically heterogeneous and a chronic and acute form is discerned. Characteristic of the chronic form is the development of cellular hepatocarcinoma. Although p-hydroxyphenylpyruvic acid (pHPPA) is used as one of the diagnostic markers of this disease, it was suggested that it is unlikely to be involved in the pathophysiology of HT1 as it is present in other disorders that does not have hepatorenal symptoms. It was the aim of this study to investigate the possible effect of pHPPA on DNA damage and repair in mammalian cells. The comet assay was used to establish the genotoxicity of pHPPA in human peripheral blood lymphocytes and isolated rat hepatocytes after their exposure to pHPPA. At first glance the damage to DNA caused by pHPPA seemed reparable in both cell types, however, after challenging the DNA repair capacity of metabolite-treated cells with treatment with H(2)O(2), a marked impairment in the DNA repair capability of these cells was observed. We suggest that the main effect of pHPPA is the long-term impairment of the DNA repair machinery rather than the direct damage to DNA and that this effect of pHPPA, together with the other characteristic metabolites, e.g., FAA and MAA, causes cellular hepatocarcinoma to develop in the chronic form of HT1.     

Stimulation of DNA synthesis and myo-inositol incorporation in mammalian cells

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material during 0–60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G₁ by amino acid starvation is not accompanied by increased incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G₁ than cells arrested by serum depletion. Inhibition of PI synthesis by δ-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0–4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G₁ and subsequent entry into S phase.     

Repair of single nucleotide DNA mismatches transfected into mammalian cells can occur by short-patch excision

Synthetic DNA linkers containing a single mismatched nucleotide (C:A) are repaired without bias at high efficiency when introduced into mammalian cells on a SV40 shuttle vector. From the pattern of repair in vectors containing multiple linkers, it appears that DNA synthesis following mismatch excision can replace a length of DNA as short as 40 nucleotides. Furthermore, results from the introduction of linker molecules containing combinations of single-strand nicks suggest that transient unsealed nicks do not drive the direction of mismatch repair in mammalian cells, as has previously been proposed.     

H-thymidine incorporation into nuclear DNA of leaf cells

The incorporation of ³H-thymidine into nuclear DNA of leaf cells of Nanthium pennsylvanicum was studied as a function of concentration and specific activity of the radioisotope. From the assessment of the average number of grains per nucleus and the percent of labeled nuclei, it was concluded that the incorporation was a linear function of concentration of the exogenous radioisotopic solution and a logarithmic function of the incubation time. Ten microcuries per milliliter on the average yielded 20% of labeled nuclei with 18 grains per nucleus. Seven-fold increase in concentration only doubled the amount of ³H-thymidine incorporated. The lamina regions near the vein incorporated a significantly greater amount of the radioisotope than the lamina region at some distance from the vein. The specific activities of 2, 3.35, 6.7 and 15.3 c/mmole had no effect upon the amount of ³H-thymidine incorporated, if the amount of microcuries of the incubation solution was the same in each activity. Considering the total number of molecules, the estimated rates of incorporation indicated that at the activity of 2 c/mmole, the system operated with about 7 times higher rates as compared with the activity of 15.3 c/mmole.     

Respective roles of pyrimidine dimer and pyrimidine (6-4) pyrimidone photoproducts in UV mutagenesis of simian virus 40 DNA in mammalian cells.

UV light induces DNA lesions which are mutagenic in mammalian cells. We used simian virus 40 tsB201 (unable to produce viral capsid at the restrictive temperature of 41 degrees C because of a point mutation in the VP1 gene) to analyze the mutagenic potency of the two major UV-induced lesions, pyrimidine dimers (Py-Py) and pyrimidine (6-4) pyrimidones [Py(6-4)Py], which are formed on the same nucleotide sites. The mutagenesis criterion was the reversion toward a wild-type growth phenotype. After UV irradiation (mainly at 254 nm), part of the DNA was treated with the photoreactivating enzyme of Escherichia coli, which monomerizes Py-Py but does not modify the Py(6-4)Py photoproduct. Higher survival and lower mutation frequency rates for the photoreactivated DNA indicated that the two lesions were lethal and mutagenic. The VP1 gene of some mutants was entirely sequenced. The mutation spectra showed that the two lesions did not induce the same mutation hot spots, although some sites were common to both. The induced mutation hot spots were not only correlated with lesion hot spots but seemed partially directed by local DNA structures.     

Repair replication in replicating and non-replicating DNA after irradiation with UV light.

Ultraviolet light induces more pyrimidine dimers and more repair replication in DNA that replicates within 2 to 3 hr of irradiation than in DNA that does not replicate during this period. This difference may be due to special conformational changes in DNA and chromatin that might be associated with semiconservative DNA replication.     

Isolation of antibiotic-producing Actinomycetes from soil samples exposed to UV light

The use of nonroutine means in isolation of microorganisms from natural substrates extended the possibilities of detecting new cultures which often appear to be producers of previously unknown antibiotics. A new procedure for isolating actinomyces of definite groups was developed. It implies preliminary exposure of soil suspensions to UV light. With the use of the procedure, 2539 strains of actinomycetes belonging to different genera were isolated. There was a marked decrease after the irradiation in isolation of cultures belonging to Streptomyces, a genus most widely distributed in nature and studied in detail while isolation of cultures belonging to other genera, promising as sources of novel antibiotics, increased. Micromonospora, Amycolatopsis and Nocardia proved to be the most stable to the effect of UV light. With the use of the procedure it is possible to increase 2-3-fold isolation of cultures belonging to Micromonospora, a genus known as a producer of many antibiotics including those used clinically.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.