The effect of hydroxyurea and trichostatin a on targeted nucleotide exchange in yeast and Mammalian cells.

Abstract

Targeted nucleotide exchange (TNE) is a process by which a synthetic DNA oligonucleotide, partially complementary to a site in a chromosomal or an episomal gene directs the reversal of a single nucleotide at a specific site. To protect against nuclease digestion, the oligonucleotide is modified with derivative linkages among the terminal bases. We have termed these molecules modified single-stranded oligonucleotides (MSOs). Current models suggest that the reaction occurs in two steps. The first, DNA pairing, involves the alignment of the MSO with the target site and its assimilation into the target helix forming a D-loop. The second phase centers around the repair of a single base mismatch formed between the MSO and its complementary strand in the D-loop. Nucleotide exchange is promoted in all likelihood by the mismatch repair system. A critical feature of successful TNE is the accessibility of the target site for the MSO and the factors that increase the dynamic nature of the chromatin that will likely increase the frequency. Here, we report that two factors, trichostatin A and hydroxyurea, elevate gene repair of a mutant hygromycin gene in Saccharomyces cerevisiae and a mutant eGFP gene in a mammalian cell line, MCF-10AT1 cells. Trichostatin A (TSA) acts by preventing the deacetylation of histones while hydroxyurea (HU) reduces the rate of replication. Both of these activities, by their very nature, create a more open configuration of the MSO into the target site.