Replication and methylation of DNA in cells of tobacco suspension culture and the effect of auxin

Abstract

After 2 min of incubation of tobacco cell culture in a medium with [3H] -- thymidine the bulk of radioactivity of newly synthesized DNA is found in short (about 5S) fragments, whereas after a prolonged incubation of the cells, i. e. 5--60 min--in long replication fragments as well. Hence DNA replication in tobacco cells occurs discretely via formation and cross-linking of Okazaki fragments. At high cell concentrations in the medium the linking of 5S fragments is suppressed. It was shown that the Okazaki fragments and other fragments of DNA replication are subjected to methylation, the DNA methylation occurring immediately after the onset of replication. The level of methylation of the 4--6S fragments is two times less than that of the linked ones; therefore replicative methylation occurs in at least two steps: at first the Okazaki fragments undergo methylation and once they are linked, an additional methylation of DNA takes place. Auxin (2,4-dichlorophenoxyacetic acid) at concentration of 5 mg per 1 of medium does not affect the ratio of the replication fragments and methylation of the Okazaki fragments, but completely inhibits the second step of replicative methylation of DNA, i. e. methylation of the linked fragments. Phytohormones can probably control the transcription of newly synthesized DNA via regulation of methylation.