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. 1975 Jun;2(6):809–819. doi: 10.1093/nar/2.6.809

Repair methylation of parental DNA in synchronized cultures of Novikoff hepatoma cells.

J K Hilliard, T W Sneider
PMCID: PMC343468  PMID: 167353

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams R. L. The relationship between synthesis and methylation of DNA in mouse fibroblasts. Biochim Biophys Acta. 1971 Dec 16;254(2):205–212. doi: 10.1016/0005-2787(71)90829-x. [DOI] [PubMed] [Google Scholar]
  2. Billen D. Methylation of the bacterial chromosome: an event at the "replication point"? J Mol Biol. 1968 Feb 14;31(3):477–486. doi: 10.1016/0022-2836(68)90422-1. [DOI] [PubMed] [Google Scholar]
  3. Burdon R. H., Adams R. L. The in vivo methylation of DNA in mouse fibroblasts. Biochim Biophys Acta. 1969 Jan 21;174(1):322–329. doi: 10.1016/0005-2787(69)90257-3. [DOI] [PubMed] [Google Scholar]
  4. CERIOTTI G. Determination of nucleic acids in animal tissues. J Biol Chem. 1955 May;214(1):59–70. [PubMed] [Google Scholar]
  5. Evans H. H., Evans T. E., Littman S. Methylation of parental and progeny DNA strands in Physarum polycephalum. J Mol Biol. 1973 Mar 15;74(4):563–572. doi: 10.1016/0022-2836(73)90047-8. [DOI] [PubMed] [Google Scholar]
  6. Kappler J. W. The kinetics of DNA methylation in cultures of a mouse adrenal cell line. J Cell Physiol. 1970 Feb;75(1):21–31. doi: 10.1002/jcp.1040750104. [DOI] [PubMed] [Google Scholar]
  7. Ryan A. M., Borek E. Methylation of DNA in ultraviolet-irradiated bacteria. Biochim Biophys Acta. 1971 Jun 30;240(2):203–214. doi: 10.1016/0005-2787(71)90659-9. [DOI] [PubMed] [Google Scholar]
  8. Scarano E., Iaccarino M., Grippo P., Winckelmans D. On methylation of DNA during development of the sea urchin embryo. J Mol Biol. 1965 Dec;14(2):603–607. doi: 10.1016/s0022-2836(65)80211-x. [DOI] [PubMed] [Google Scholar]
  9. Schein A., Berdahl B. J., Low M., Borek E. Deficiency of the DNA of Micrococcus radiodurans in methyladenine and methylcytosine. Biochim Biophys Acta. 1972 Jul 20;272(3):481–485. doi: 10.1016/0005-2787(72)90400-5. [DOI] [PubMed] [Google Scholar]
  10. Schneiderman M. H., Billen D. Methylation rapidly reannealing DNA during the cell cycle of Chinese hamster cells. Biochim Biophys Acta. 1973 May 18;308(3):352–360. doi: 10.1016/0005-2787(73)90327-4. [DOI] [PubMed] [Google Scholar]
  11. Sneider T. W. Methylation of mammalian deoxyribonucleic acid. II. The distribution of 5-methylcytosine in pyrimidine deoxyribonucleotide clusters in Novikoff hepatoma cell deoxyribonucleic acid. J Biol Chem. 1971 Aug 10;246(15):4774–4783. [PubMed] [Google Scholar]
  12. Sneider T. W., Potter V. R. Methylation of mammalian DNA: studies on Novikoff hepatoma cells in tissue culture. J Mol Biol. 1969 Jun 14;42(2):271–284. doi: 10.1016/0022-2836(69)90043-6. [DOI] [PubMed] [Google Scholar]
  13. Turkington R. W., Spielvogel R. L. Methylation of deoxyribonucleic acid during hormonal stimulation of mammary cells in vitro. J Biol Chem. 1971 Jun 25;246(12):3835–3840. [PubMed] [Google Scholar]
  14. Walters R. A., Tobey R. A., Ratliff R. L. Cell-cycle-dependent variations of deoxyribonucleoside triphosphate pools in Chinese hamster cells. Biochim Biophys Acta. 1973 Sep 7;319(3):336–347. doi: 10.1016/0005-2787(73)90173-1. [DOI] [PubMed] [Google Scholar]

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