Gene Targeting and “Hit-and-Run” Transformation

Abstract

Gene targeting is a genetic technique that uses homologous recombination to change an endogenous gene. Although many studies identified key genetic requirements for gene targeting and it is being used in experiments every day to delete a gene, add a gene, or replace a gene, many aspects of the mechanism is still unknown. I have been studying one phenomenon that results from gene targeting, referred as "hit-and-run" transformation, initially described by Kraus, Leung and Haber (2001) for budding yeast. In Kraus's paper, it has been found that some cells make plasmid-like circular DNA during intended gene targeting. For this to occur, the targeting fragment had to recombine with the target locus but somehow ended up by copying adjacent sequences, including a nearby origin of replication, and then became an autonomously replicating circle. How such circles are formed or what mechanism drives the formation of circular DNA is not well understood. In order to study more about the "hit-and-run" events, I examined gene targeting in Saccharomyces cerevisiae, in which an ade2::URA3 fragment was used to delete the wild type ADE2 gene. When I performed regular gene targeting transformation using the ade2::URA3 PCR product, selecting for URA3, I observed three different results: (1) about 69% were stable ADE-URA+ transformants that represent normal successful gene targeting; (2) about 25% were ADE+URA+ events where the URA3 sequences apparently integrated at another location by illegitimate integration and (3) about 6% were ADE+URA+ transformants in which the URA3 marker could be lost by selection on 5-FOA, which is the result expected for a “hit-and run” event. I went further and tried to examine the FOA-resistant isolates that resulted from ade2::URA3 gene targeting, and using inverse PCR, I found out that some of these hit-and-run events were indeed circles. The sizes of the circles varied among the isolates. In addition, the previous paper by Kraus et al. concluded that the “hit-and-run” event seemed to be Break-induced replication-dependent that can have extended DNA synthesis without crossing-over (2001). In order to test this hypothesis, I created a mutant with pol32Δ which is required for BIR and performed ade2::URA3 gene targeting and got the results: about 1% were stable Ade-Ura+ transformants; about 99% were illegitimate ADE+URA+ FOA-sensitive transformants; and 0% no hit-and-run event.