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[Methylation of newly synthesized DNA in mouse fibroblast culture]
Abstract:
After a 10 min- or more prolonged incubation of transformed mouse fibroblasts (L.-cells) with [3H]-thymidine or [3H-methyl]-methionine and a subsequent centrifugation of cell lysates in an alkaline sucrose gradient the DNA radioactivity is detected in long (28, 33 and 45S) and short (5, 13 and 18S) fragments. An increase in cell concentration in the cultural layer results in inhibition of 5S fragments linkage rather than in inhibition of their synthesis. The blocking of the Okazaki fragment linkage may be regarded as one of the inhibitory molecular mechanisms of cell depletion. Both in the case of normal and suppressed (by 99%) replication by arabofuranosylcytosine [3H]-thymidine and [3H-5-methyl] cytosine are detected in the Okazaki fragments (5S) as well as in some discrete lower molecular weight fractions (lesser than 5S) of newly synthesized DNA. Thus, replicative methylation of DNA in the fibroblasts occurs in the replicative fork during DNA synthesis and the functioning DNA methylase is an indispensable component of the replicative complex. The methylation of Okazaki fragments is non-chaotic and has a specificity other than that of total DNA. This may be due to the multiplicity and different specificity of nuclear DNA-methylases. Thus, there exist in animal cells replicative and post-replicative methylation of DNA, which may differ in the nature of substrates and enzymes, in specificity of recognizable sequences and in their functional significanse.
Insights
DNA methylation occurs during replication in fibroblasts, with Okazaki fragments methylated specifically. This suggests distinct replicative and post-replicative methylation processes in animal cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA replication involves the synthesis of short DNA fragments known as Okazaki fragments.
- DNA methylation is a crucial epigenetic modification regulating gene expression and DNA replication.
- Understanding the timing and mechanisms of DNA methylation during replication is essential for comprehending genome stability and cellular processes.
Purpose of the Study:
- To investigate the role and timing of DNA methylation during the replication of DNA in transformed mouse fibroblasts.
- To determine if DNA methylation occurs concurrently with DNA synthesis at the replication fork.
- To explore the specificity and potential differences between replicative and post-replicative DNA methylation.
Main Methods:
- Incubation of transformed mouse fibroblasts (L-cells) with radiolabeled nucleotides ([3H]-thymidine, [3H-methyl]-methionine, [3H-5-methyl] cytosine).
- Separation of newly synthesized DNA fragments using alkaline sucrose gradient centrifugation.
- Detection and analysis of radioactivity in different molecular weight DNA fractions.
Main Results:
- Newly synthesized DNA was found in both long and short fragments, including Okazaki fragments (5S).
- Radiolabeled precursors were incorporated into Okazaki fragments and smaller DNA fractions during both normal and suppressed replication.
- Increased cell concentration inhibited the linkage of 5S fragments, suggesting a role in cell depletion.
- Methylation of Okazaki fragments exhibited specificity distinct from total DNA, implying multiple DNA methylases.
Conclusions:
- DNA methylation occurs at the replication fork during DNA synthesis in fibroblasts, with DNA methylase being integral to the replication complex.
- Okazaki fragment methylation is specific and suggests the involvement of multiple nuclear DNA-methylases with varying specificities.
- Animal cells exhibit distinct replicative and post-replicative DNA methylation processes, differing in substrates, enzymes, recognition sequences, and functional significance.