Studies on the Influence of Cytosine Methylation on DNA Recombination and End-joining in Mammalian Cells

Abstract

To test the influence of cytosine methylation on homologous recombination and the rejoining of DNA double strand breaks in mammalian cells, we developed a sensitive and quantitative assay system using extrachromosomal substrates. First, methylation was introduced into substrates in vitro with the prokaryotic SssI methylase, which specifically methylates the C-5 position of cytosine bases within CpG dinucleotides, mimicking the mammalian DNA methyltransferase. Next, methylated substrates were incubated in mammalian cells for a sufficient length of time to recombine or rejoin prior to substrate recovery. Results from bacterial transformation of the substrates and from direct Southern analysis demonstrate that cytosine methylation has no detectable effect on either DNA end-joining or homologous recombination. Thus, the components of the protein machinery involved in these complex processes are unaffected by the major DNA modification in mammalian cells. These results leave open the possibility that methylation may modulate the accessibility of these components to chromosomal DNA by altering local chromatin structure.

Suppression of crossing-over by DNA methylation in Ascobolus

“…As for homologous recombination, a general process that takes place in any living organism, no direct evidence for an inhibitory effect of methylation has been established so far. Attempts to demonstrate such an effect, using extrachromosomal plasmids, have been unsuccessful (Puchta et al 1992;Liang and Jasin 1995).By using MIP to methylate a 7.5-kb chromosomal interval encompassing a meiotic recombination hot spot of A. immersus, we show here that DNA methylation reduces by several hundredfold the frequency of crossingover within this interval. We provide additional experimental evidence indicating that this effect cannot be simply accounted for by an increased resistance of methylated DNA to meiotic endonucleases.…”

“…As for homologous recombination, a general process that takes place in any living organism, no direct evidence for an inhibitory effect of methylation has been established so far. Attempts to demonstrate such an effect, using extrachromosomal plasmids, have been unsuccessful (Puchta et al 1992;Liang and Jasin 1995).…”

Eukaryotic DNA methylation as an evolutionary device

“…(78) No suppressing effect of methylation on somatic recombination in mammalian cells could be detected in transfection experiments with methylated plasmids, however. (79) Given that in these experiments recombination might have arisen before chromatin assembly, a lack of effect may not be relevant, since the suppressing effect of methylation is likely to require a chromatin context. (75,79) Furthermore, the increased mutation rate and the types of mutations observed in hypomethylated mouse ES cells add significantly to the body of evidence in favor of an inhibitory effect of methylation on somatic recombination in mammals.…”

Genome-wide demethylation promotes triplet repeat instability independently of homologous recombination

“…CpG-methylated and unmethylated DNA constructs, when transfected into COS1 monkey cells, gave identical frequencies of extrachromosomal recombination in an assay designed to detect single-strand annealing, one kind of HR [19]. Similarly, 5-aza-CdR treatment of HeLa human cells that were modified to carry a chromosomal recombination substrate designed to detect conversion events did not increase the frequency of HR [20].…”