Abstract
Previous studies have shown both hypermethylation and late replication of DNA sequences to be associated with gene inactivity. To determine whether there is a causal relationship between patterns of DNA methylation and replication timing during S phase, we have examined the timing of replication of the inactive, hypermethylated metallothionein(MT) I andII genes in synchronized, cadmium-sensitive (Cds) CHO cells. The time of S-phase replication of theMT genes was ascertained by (1) determining the period of S phase wherein cadmium-resistant (Cdr) cells could be induced with highest frequency by pulse treatment of synchronized Cds cells with the hypomethylating drug 5-azacytidine (5-aza-CR), and (2) by analyzing Southern blots of density fractionated DNAs isolated from synchronized cells pulse-labeled with BrdU during different intervals after release from hydroxyurea blockade. Southern filter hybridization analyses demonstrated replication of bothMTI andII gene sequences within the first half of S phase. Consistent with this result, phenotypic conversion of Cds to Cdr was maximal immediately after hydroxyurea release and decreased abruptly within three hours. The replication of inactive hypermethylatedMT genes in early S phase argues that transcriptional inactivity and gene-specific hypermethylation are not sufficient conditions for late DNA replication.
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Stallings, R.L., Crawford, B.D., Tobey, R.A. et al. 5-Azacytidine-induced conversion to cadmium resistance correlates with early S phase replication of inactive metallothionein genes in synchronized CHO cells. Somat Cell Mol Genet 12, 423–432 (1986). https://doi.org/10.1007/BF01539913
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DOI: https://doi.org/10.1007/BF01539913