The homology-dependent repair of DSBs usually occurs by a conservative gene conversion mechanism, preventing extensive loss of heterozygosity (LOH) or chromosome rearrangements.  Breaks that present only one end for repair, for example at eroded uncapped telomeres or when homology is limited to one side of the DSB, are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). As BIR from one of the two ends of a DSB would result in extensive LOH, this suggests BIR is suppressed when DSBs have two ends in order for repair to occur by a more conservative HR mechanism. BIR and a related mechanism, fork stalling and template switching (FoSTeS), are thought to be responsible for many of the genome rearrangements that give rise to non-reciprocal translocations and copy number variation associated with human disease.

We have developed several assays to study BIR in yeast. Our early work utilized a plasmid-based chromosome fragmentation vector (CFV), but we have since developed a chromosomal system to monitor BIR. In this system, an HO endonuclease recognition site (HOcs) is integrated 32 kb from the left telomere of chromosome (Chr) V in a strain expressing a galactose-inducible HO gene. The HOcs is directly adjacent to a 3’ truncated LYS2 gene (“lys”) on the centromeric side, and a KanMX gene providing resistance to gentamycin (G418) on the telomeric side. A 5’ truncated LYS2 gene (“ys2”) inserted 70 kb from the left telomere of Chr XI provides homology for repair. These two lys2 fragments share 2.2 kb homology, and are non-functional before recombination since they lack the complete ORF. After transferring cells to galactose-containing medium to induce the DSB, the centromeric side of the break containing “lys” invades the homologous “ys2” site and copies to the end of donor Chr XI by BIR. This heals the break and creates a functional LYS2 gene on the recipient chromosome. The distal end of Chr V contains no essential genes and is lost due to lack of available homology. More than 99% of cells that grow on galactose-containing medium are Lys+ and have lost the KanMX marker located centromere-distal to the DSB on the recipient chromosome. BIR require the same factors as gene conversion suggesting the initial strand invasion step is catalyzed by a common set of proteins. In contrast to gene conversion repair, BIR requires the non-essential subunit of the DNA polymerase δ complex, Pol32. Our studies of BIR revealed that the initial strand invasion intermediate is unstable and can undergo multiple cycles of dissociation and strand invasion, sometimes resulting in the formation of non-reciprocal translocations.