DNA repair in pollen: Range of mutagens inducing repair, effect of replication inhibitors and changes in thymidine nucleotide metabolism during repair

Abstract

Pollen of Petunia hybrida carry out DNA repair during the first two hours of germination when certain mutagens are included in the germination medium. This repair, detected readily as unscheduled DNA synthesis, since there is no replicative DNA synthesis in Petunia pollen, can be induced by the chemical mutagens N-methyl-N′-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, azaserine and methyl methanesulphonate. These compounds are all considered to be capable of direct covalent interaction with DNA. Mutagens requiring metabolic activation before interaction with DNA did not induce DNA repair synthesis in pollen. The practice of solubilizing water-insoluble chemical mutagens with dimethyl sulphoxide did not prove practical, due to the extremely harmful effects of dimethyl sulphoxide on pollen. Pretreatment of pollen before germination with pure ether, however, had no harmful effect on either repair or pollen germination. Therefore water-insoluble, ether-soluble mutagens were tested by pretreatment of the pollen with mutagens in ether solution. By this means it was shown that the ‘direct-acting’ mutagen, diethyl sulphate, would also bring about unscheduled DNA synthesis in pollen, while 2-acetylaminofluorence and dimethyl-p-aminobenzene, both requiring metabolic activation, did not do so. Inhibitors of DNA replicative synthesis, hydroxyurea, azaserine, azauridine and fluorodeoxyuridine did not inhibit unscheduled DNA synthesis brought about by N-methyl-N′-nitro-N-nitrosoguanidine. On the contrary, these compounds stimulated repair synthesis to varying degrees, hydroxyurea having the greatest effect. Pollen uptake of 3H-thymidine and the amount of radioactive label subsequently appearing in dTMP and dTDP+dTTP was increased by 4-nitroquinoline-1-oxide. Partial inhibition of these increases and of 4-nitroquinoline-1-oxide induced repair synthesis by 3′,5′-cyclic AMP suggested that thymidine:AMP phosphotransferase rather than thymidine kinase was responsible for thymidine phosphorylation in pollen. Enzyme assays on pollen extracts confirmed this.