Repair methylation of parental DNA in synchronized cultures of Novikoff hepatoma cells.
Parental and filial DNA strands were isolated from a Novikoff rat hepatoma cell line, synchronized by S-phase arrest with excess thymidine, that had completed up to one round of DNA replication in the presence of (14-C-methyl)methionine and (6-3-H) bromodeoxyuridine. Both strands were methylated, the proportion of total methyl label in parental DNA increasing slightly with time in S-phase. The studies were repeated with (14-C-methyl)methionine and (3-H)deoxycytidine to determine if parental methylation occurred on extant or repair-inserted cytosine residues. Both (14-C) and (3-H) were found in parental DNA. The (14-C)/(3-H) ration of parental DNA-5-methylcytosine was about twice that in filial DNA while the (3-H) data showed twice the concentration of 5-methylcytosine in parental compared to filial DNA. Thus parental methylation occurred on repair-inserted cytosine residues and resulted in overmethylation. That the DNA damage and repair was due to 5-phase arrest was shown by repeating the studies using a sequential mitotic-G1 arrest method. With this method little (14-C) or (3-H) was found in parental DNA. We conclude that S-phase arrest leads to DNA damage and repair with subsequent overmethylation of repair-inserted cytosines; that sequential mitotic-G1 arrest minimizes DNA damage; 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.
D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D003841:Deoxycytidine / Q000378:metabolism; D008113:Liver Neoplasms; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D009374:Neoplasms, Experimental / Q000378:metabolism; D051381:Rats; D013936:Thymidine / Q000378:metabolism
D003841:Deoxycytidine; D004247:DNA; D008715:Methionine; D001973:Bromodeoxyuridine; D013936:Thymidine