Caffeine affects the level of gene repair in mammalian cells; implications for a role of DNA replication in the correction of single base mutations

Single stranded DNA oligonucleotides have been used to direct the correction of point mutations in mammalian cells. The introduction of a single-stranded oligonucleotide sets in motion a cascade of events that eventually leads to a stalling of DNA replication forks, which enables the oligo-mediated correction event to occur more readily. This is due to the fact that cells in S phase are more amenable to gene correction and a role for DNA replication in the gene repair reaction has been established. Here, we develop a mechanistic view of how cell cycle arrest might influence the gene repair reaction using the radiosensitizing agent, caffeine, as a tool. First, we demonstrate that ATM activation is enhanced and sustained by caffeine then we identify multiple roles for caffeine in the modulation of the gene repair reaction. Finally, we show that caffeine-treated cells retain the oligonucleotides for longer periods of time, perhaps enabling the oligo to assimilate into the target site more readily. Our results may define ATM activation upon the entry of the oligonucleotide as a likely block to the proliferation of corrected cells.