Studies on the influence of cytosine methylation on DNA recombination and end-joining in mammalian cells.
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.
Liang
F
F
Cell Biology and Genetics Program, Sloan-Kettering Institute, New York, New York, USA.
Jasin
M
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D003596:Cytosine / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D015257:DNA-Cytosine Methylases; D005821:Genetic Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D014162:Transfection
D003596:Cytosine; D004247:DNA; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases