384. Double-Stranded DNA Breaks Stimulate the Frequency of Targeted Gene Repair in Mammalian Cells

Abstract

We have reported that double-stranded breaks in DNA stimulate the gene repair activity of the homologous recombination pathway. This induction also leads to a stalling of DNA replication thereby slowing the cell cycle phase in which the gene repair reaction occurs most often. DNA cleavage was induced initially by using anticancer drugs like etoposide (VP16), camptothecin (CPT) or chemicals like hydroxyurea (HU) and methymethanesulfonate (MMS). The last of these agents, MMS, is an alkyaltion agent capable of inducing DNA lesions and subsequent single and double strand breaks (DSB). This damage activates the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways, a response that has been shown to increase the frequency of oligonucleotide-directed gene repair. We show in DLD-1 cells that the conversion frequency of a mutant base pair to wild-type in an integrated enhanced green fluorescent protein gene is increased by pretreatment with MMS. This stimulation is dose-dependent and correlates to the level of DSB induced by MMS treatment. These DSBs result in Rad51p nuclear-relocalization and foci formation, a specific marker for HR activation. Additionally the cells exhibit cell cycle delay in S phase due to the stalling of replication forks. Our data suggest that MMS-induced DNA damage elicits a cellular response that stimulates gene repair in mammalian cells.