Repair methylation of parental DNA In synchronized cultures of Novikoff hepatoma cells

Abstract

Parantal and filial DNA strands ware isolated from a Novikaff rat hapatoma call line, aynchronized by S-phase arrest with excess thymidine, that had completed up to one round of DNA replication in the prasanca of (¹⁴C-methyl)methionine and (6-3H)bromodeoxyurid ina. Both strands ware methylated, the proportion of total methyl label in parantal DNA Increasing slightly with time in Sphaae. The studies were repeated with (¹⁴C-methyl)methionine and (³H)deoxycytldine to determine if parental mathylation occurred on axtant or repair-inserted cytosine residues. Both (¹⁴C) and (³H) mere found in parental DNA. The (¹⁴C)/(³H) ratio of parental DNA-5-methylcytosine was about twice that in filial DNA while the (³H) data showed twice the concentration of 5-methylcytosine in parental compared to filial ONA. Thus parental methylation occurred on repair-inserted cytosine residues and resulted in ovarmethylation. That the DNA damage and repair was due to S-phase arrast was shown by repeating the studies using a sequential mitoticd arrest method. With this method little (¹⁴c) or (³H) was found in parental DNA. Me conclude that S-phase arrest leads to DNA damage and repair with subsequent overmethylation of repairinserted cytoainasi that sequential mitotic-G₁ arrest minimizes DNA damagel and, that the latter technique, suitable for synchronization of large quantities of cells, may prove useful in relatively artifact-free studies of eukaryotic DNA replication.