Akira Sono

A meiotic DNA polymerase from Coprinus cinereus: further purification and characterization

A meiotic DNA polymerase that is present at a high level of activity in meiotic cells of a basidiomycete, Coprinus cinereus, was purified to near homogeneity using synthetic RNA homopolymer [poly(C)] cellulose column chromatography. This report presents the first extensive purification and characterization of any eukaryotic DNA polymerase having a role in meiosis. This enzyme is a single polypeptide with a molecular mass of 65,000. Activity in this enzyme requires magnesium ions and occurs at an optimal pH of 7.5. It is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmaleimide and can use poly(C)/oligo(dG)12–18 as a template-primer. Polymerase activity can be found only in cells at meiotic prophase, even though the enzyme has been identified in somatic cells in an inactive state using immunoblot analysis. Its distinctive distribution makes possible a genetic and biochemical analysis of functional role of a meiotic DNA polymerase in meiotic recombination, repair and synthesis.

Inhibition of protein synthesis antagonizes induction of sister-chromatid exchanges by exogenous agents.

Cycloheximide (CH) and puromycin (PM)strongly antagonize induction of sister-chromatid exchanges (SCEs) by exogenous agents regardless of the mechanism for initiating damage. 5-Bromodeoxyuridine (BUdR) substitution was used to monitor SCEs, but the background level of BUdR-induced SCEs was unaffected by the presence of protein inhibitors. Antagonism between DNA-damaging agents and protein inhibitors was strongest in euchromatic regions. Possible relationships between SCE formation and the mechanism of antagonism by protein inhibitors are discussed.

Antagonization by cycloheximide of ethyl methanesulfonate-induced 6-thioguanine-resistant mutation and sister-chromatid exchanges

We studied the effect of cycloheximide (CH) on the induction of mutation and sister-chromatid exchanges (SCEs) in ethyl methanesulfonate (EMS)-treated Chinese hamster cells. At 10(-6)M, CH strongly antagonized the induction of mutation and SCEs and cell survival increased. This suggests that protein synthesis is essential for the induction of mutation as well as SCEs. Results of experiments in which CH treatment preceded or followed exposure to mutagens were similar with respect to the response curves obtained for mutation and SCEs.

The influence of a protein synthesis inhibitor on sister-chromatid exchange in the plant Vicia faba

Cycloheximide strongly antagonizes the induction of sister-chromatid exchange by mitomycin C in Vicia faba root tips. This behavior is analogous to that previously observed in mammalian cells (Sono and Sakaguchi, 1981) and suggests that newly synthesized protein is also required for recombination between sister DNA molecules in plants. Conversely hydroxyurea is shown to increase the frequency of both spontaneous and induced sister-chromatid exchange. Based on these results, possible mechanisms underlying sister-chromatid exchange formation in plants are discussed with special emphasis on the absence of DNA polymerase beta in somatic tissues.

Isolation of Chinese hamster cells hyposensitive to ethyl methanesulfonate and their characterization in induction and antagonization of sister-chromatid exchanges.

To better understand the molecular events involved in the formation of SCEs and mutations, 3 clonal sublines were established from a Chinese hamster cell line, Don D-6, on the basis of hyposensitivity to killing by ethyl methanesulfonate. These sublines, referred to as T80-4, T80-6 and T80-7, exhibited greatly altered karyotypes and altered frequencies of SCEs and mutations. These sublines were also less sensitive to chemical mutagens and ultraviolet irradiation as indicated by reduced frequencies of SCEs. This lower sensitivity was not due to differences in the rate of cell proliferation or to a cellular adaptive response. The rate of chemically- or UV-induced SCEs in these sublines did not always relate to cellular ploidy levels. The recent observation that, under constant experimental conditions, the rate of SCEs is a function of cellular ploidy level was not observed for these sublines. Thus, there appeared to be a modification in the intrinsic process of SCE formation. We reported previously that protein synthesis is essential for the induction of mutation as well as for SCE in Don D-6. However, the ability of cycloheximide (CH) to antagonize EMS-induced SCEs and mutations in the T80-7 subline was much lower than in the parental cells or in other sublines. The cells of this subline might contain a defective protein or a modification of the process for which the protein is required.

Isolation and characterization of Chinese hamster cell line resistant to monofunctional alkylating agents

A clonal derivative of a Chinese hamster Don D-6 cell line resistant to methyl methane sulfonate (MMS) has been isolated following mutagenesis by ethyl methane sulfonate (EMS). The clone, designated as MMSr-1, exhibited high resistance to killing by the monofunctional alkylating agents MMS and EMS. This characteristic had not been acquired by a transient adaptation to the alkylating agents but was found to be a stable heritable trait. MMSr-1 was more sensitive to high-molecular-weight chemicals, such as colchicine and puromycin, than Don D-6. Both MMSr-1 and its parental cells showed the same ability to take up radioactive MMS. The resistance of MMSr-1 appears not to be due to altered uptake of MMS. The resistance was accompanied by low chromosomal aberration and sister chromatid exchange (SCE) induction but not by mutability. Protein synthesis inhibitors such as cycloheximide and puromycin reduced the resistance to the same level as that in Don D-6. SCE induction by MMS in this clone was not antagonized by the protein synthesis inhibitors, whereas mutagenesis was reversed to the normal parental cell level by these inhibitors. Aphidicolin, a DNA-synthesis inhibitor, exhibited no such effects. These results suggest that MMSr-1 might have modified repair capacity, which can be normalized by treatment with the protein-synthesis inhibitors, for lethal DNA damage by monofunctional alkylating agents, and that SCE formation by the alkylating agents is closely correlated with chromosomal aberration and cell lethality.