1056. Suppression and Enhancement of Gene Repair by the Modulation of ATM Function in Mammalian Cells

Abstract

Targeted gene repair utilizes oligonucleotides to direct single base alterations in the genomes of mammalian cells. These vectors are designed to hybridize to the site of interest and to create a single mismatch with the mutant base. Although the mechanism of the nucleotide exchange phase of the reaction remains to be fully elucidated, evidence suggests that the induction of homologous recombination (HR) in response to DNA damage elevates DNA pairing activity and results in increased levels of gene repair. The HR pathway is controlled by the activation of the cell cycle checkpoint regulator, Ataxia-Telangiectasia mutated (ATM), which is induced by either DNA damage or by the presence of single stranded DNA. The radio-sensitizer, caffeine, has previously been shown to inhibit ATM kinase activity, by interfering with the phosphorylation of downstream substrates. When caffeine was added to a gene repair reaction prior to the introduction of the oligonucleotide, the frequency of correction was reduced, reinforcing the importance of HR in gene repair. If, however, caffeine was added after the oligonucleotide, the correction frequency was enhanced suggesting a dual functionality for caffeine in the gene repair reaction. Thus, we utilize caffeine as a reagent and a tool to understand the regulation of this reaction in greater detail. ATM activation in cells exposed to 4mM caffeine was examined following oligonucleotide delivery. At 18 and 24 hours, the level of ATM activation was increased significantly and even at 48 hours post-delivery, when ATM activation form the introduction of the ssDNA vector disappears, activated ATM was still quite discernable in caffeine treated cells. This sustained ATM activation could be explained by a concurrent effect of caffeine on the gene repair reaction; the oligonucleotide is retained by the cell for a longer period than in untreated cells. At 16 hours post electroporation, both cell populations exhibited about a 70% uptake of the oligonucleotide (as measured by the fluorescence of a FAM conjugated oligonucleotide). However, between 16 and 48 hours, the non-treated cells exhibit a steep drop in oligonucleotide levels, as compared to the caffeine treated cells. This suggests that the oligonucleotide activates ATM in all cells but in cells treated with caffeine, it reactivates ATM through an extended cellular half-life. The use of caffeine as a tool for studying the regulatory pathways of gene repair lead us to conclude that the activation of ATM is a critical step in promoting successful gene correction. On one hand ATM is required to induce HR which catalyses the first phase of gene repair (DNA pairing), but on the other hand, activated ATM can block the replication of corrected cells affecting the overall frequency of the reaction. We are now modulating the levels of ATM so that the balance can be tipped in favor of more robust levels of gene repair.