385. Gene Correction in the Absence of DNA Repair: A Universal Model Involving Homologous Pairing and DNA Replication

Abstract

Our laboratory has been studying the mechanism and regulation of targeted gene repair in mammalian cells. Using a robust cell line where the genomic correction frequencies average 1%, we have been able to modulate the process of DNA replication and observe its effect on the gene repair reaction. In our hands, gene correction occurs predominantly in S phase cells especially when the movement of the replication fork is delayed. Reminiscent of its role in homeologous recombination, several labs, including ours, have observed that the mismatch repair pathway appears to interfere with gene repair activity. Taking all of these observations into consideration, we now propose a universal model for gene repair in which the oligonucleotide becomes incorporated into the newly synthesized daughter strand at or near a stalled replication fork. Assimilation of the oligonucleotide creates a single mismatched base pair which, in the case of a MMR-deficient cell line, is tolerated through a single round of replication. During a second round, the replication machinery uses the oligonucleotide as the parental template and synthesizes the second strand of a fully converted allele. This model explains a multitude of unrelated observations that surround the variability of this technique. The integrated model of gene repair will be presented with examples from yeast and mammalian cells that strongly support its validity.