Studies on the influence of cytosine methylation on DNA recombination and end-joining in mammalian cells.

Feng Liang,Maria Jasin

doi:10.1074/jbc.270.40.23838

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.

Highlights

Two key processes in the maintenance of genomic integrity in mammalian cells are DNA end-joining, a nonhomologous process in which DNA breaks are rejoined, and homologous recombination
A Sensitive Assay for Extrachromosomal Homologous Recombination—To determine whether cytosine methylation influences the efficiency of extrachromosomal homologous recombination in mammalian cells, we developed a sensitive and quantitative assay based on bacterial transformation
These results demonstrate that kanamycin resistance (KanR) is a result of homologous recombination between M5neo and M3neo in COS1 cells and, that extrachromosomal recombination is not affected by CpG methylation

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructions and Methylation Reactions—DNA manipulations were performed according to standard procedures [18]. Plasmid Mneo was constructed by cloning the 1206-bp HindIII/BamHI Tn5 neo gene fragment from pSV2neoM7 into HindIII/BamHI-cleaved pUC19. Plasmid M3neo was generated by ligating the 673-bp PstI/ BamHI neo gene fragment of pSV2neoM7 into PstI/BamHI-digested pUC19. Bacteria (40 ␮l) were mixed with 0.5 ␮l of DNA and electroporated in a 0.1-cm cuvette using a Bio-Rad gene pulser with a setting of 1.8 kV They were placed in 1 ml of SOC medium for a 1-h incubation at 37 °C prior to plating on ampicillin- or kanamycin-containing plates. Cells were washed twice in phosphate-buffered saline immediately after electroporation to remove untransfected DNA and incubated in tissue culture medium at 37 °C. To recover transfected DNA, cells were harvested after 4-h incubation They were trypsinized and washed three times in phosphate-buffered saline to remove any remaining untransfected DNA. Southern analysis was performed using 1–2 ␮l of DNA according to standard procedures [18]

RESULTS

Cytosine Methylation and Recombination

Mneo ϩ

DISCUSSION