FIG. 6.

Gene targeting. (A) When yeast cells are transformed with a replicating plasmid (ARS plasmid) containing a double-strand break or gap within a region of the plasmid with homology to the yeast genome, the break or gap is repaired by gene conversion from the chromosomal locus. The plasmid remains episomal if gene conversion without an associated crossover occurs but is integrated into the genome if conversion is associated with crossing over. If the plasmid contains a CEN sequence, only conversion events can give rise to viable transformants. If the plasmid contains no origin of replication (ARS), only integration events are observed. (B) Ends-out gene targeting refers to replacement of chromosomal sequences with sequences present on a linear DNA fragment introduced into cells by transformation. Gene targeting is thought to occur by invasion of the two ends into the chromosomal locus followed by resolution of the resulting Holliday junctions. Extensive DNA synthesis could be primed from the invading 3′ ends prior to Holliday junction resolution, or resolution could occur by replication to the end of the chromosome. Gene targeting could also result from integration of a single strand of the targeting fragment followed by trimming the D-loop and mismatch repair.