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

S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line.

Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA