STUDIES ON THE INFLUENCE OF CYTOSINE METHYLATION ON DNA RECOMBINATIONAND END-JOINING IN MAMMALIAN-CELLS

To test the influence of cytosine methylation on homologous recombinat ion and the rejoining of DNA double strand breaks in mammalian cells, we developed a sensitive and quantitative assay system using extrachro mosomal substrates. First, methylation was introduced into substrates in vitro with the prokaryotic SssI methylase, which specifically methy lates the C-5 position of cytosine bases within CpG dinucleotides, mim icking the mammalian DNA methyltransferase. Next, methylated substrate s were incubated in mammalian cells for a sufficient length of time to recombine or rejoin prior to substrate recovery. Results from bacteri al transformation of the substrates and from direct Southern analysis demonstrate that cytosine methylation has no detectable effect on eith er DNA end-joining or homologous recombination. Thus, the components o f the protein machinery involved in these complex processes are unaffe cted by the major DNA modification in mammalian cells. These results l eave open the possibility that methylation may modulate the accessibil ity of these components to chromosomal DNA by altering local chromatin structure.