Organization, replication and modification of the human genome: Synthesis and methylation of palindromic, repeated and unique hela nDNA sequences during the S-phase

Abstract

At least five supermethylated nDNA families have been found in HeLa cells. The concentration of 5-methylcytosine increases on palindromes from 1 to 5 hours during the nDNA duplicative phase, decreasing again at the 6th hour of S. The early S-phase involves the accumulation of 5-methylcytosines on the nDNA sequences reassociating near a Cot = 3 x 10(-1). Late S involves increment of this base on the sequences which reassociate near a Cot = 9 x 10(-1) and Cot = 7 x 10(1). The unique sequences show a moderate methylation from 3 to 6 hours of S. This information shows that genes are methylated with an order during the S-phase in a system in which nDNA organization, modification and replication appear to be severely subordinated one to another. The possible role of methylation of foldback nDNA in regulation of transcription during the eukaryotic cell life cycle is discussed.

In vitro methylation of total and foldback DNAs in normal and virus‐transformed cells

“…We have shown elsewhere for HeLa cells that palindromes are methylated during the S-phase in parallel with methylation of total DNA, while their methylation pattern is much higher than that of total DNA [5]. This is strikingly confirmed by the present study which involved other cell species.…”

“…Then, as expected, the kinetics of reassociation showed that the h~omethylated DNA is mainly made up of unique sequences, while the hypermethylated DNA is made up of repetitive and palindromic sequences IS]. The palindromic sequences reach a specific methylation, during the S-phase, which is much higher than that of the repetitive sequences [5]. However, a decrease of transcription corresponds, in the Sphase, to the hypermethylation of this DNA class and of total DNA [5-73. This investigation shows increased methylation of total and palindromic DNAs of transformed cells relative to untransformed cells.…”

“…This partially purified DNA fraction was denatured again for 10 min at 100°C and immediately re-chromatographed on hydroxyapatite. In this case, the C,,t value was estimated to be below 0.001, while rechromatography reduced the yield of DNA to about one third, i.e., to about 5% of the total genome [5,12]. Unlike this fraction, which contained inverted repeats of the type ABC.. .…”

“…The maximal rates of methylation for NRK and 3T3 nuclei correspond to 15 000 and 7000 dpm/A, respectively, while those for RSV-NRK and SV40-3T3 nuclei are 21500 and 18200 dpm/A, respectively ( fig.le,f). These differences are even more significant considering the fact that palindromes show in general a specific methylation pattern which is higher than that of the total DNA [5]. In other words, palindromes of virus-transformed cells are hypermethylated to a greater extent with respect to the total DNA fraction while the rate of formation of 5-methylcytosines on DNA definitely appears to be a function of the culture growth cycle and the cell species.…”

“…After dialysis and deionization on Sephadex G-25, the DNA fragments obtained were estimated to be about 600 nucleotides long by sedimentation in an alkaline sucrose gradient, using DNA length standards [5]. The double helix state of these fragments was checked through T, analysis.…”