We have developed an assay to study chromosomal rearrangements
associated with DNA double-strand break (DSB) repair in haploid yeast
cells. In this assay, an HO endonuclease recognition sequence was
inserted into an actin intron that was engineered into the URA3
locus. After induction of HO endonuclease, survivors that have become
FOA resistant were analyzed to determine how the HO induced DSBs had
been repaired. We have carried out this analysis with a set of
isogenic strains which were either WT, rad52, ku80, or
rad52ku80. We have observed that both survival and frequency of
chromosomal rearrangements are strongly dependent on the genetic
background. In the presence of a single HO recognition sequence, we
observed a variety of nonhomologous recombinational repair events
including large scale deletions, Ty1 cDNA and mitochondrial DNA
insertions, pericentric inversions, and reciprocal
translocations. When we analyzed repair events in strains containing
two HO recognition sequences on different chromosomes, most uracil
auxotrophs resulted from reciprocal rejoining events between the two
chromosomes. Analysis of the junctional sequences at breakpoints
demonstrated the presence of multiple mechanisms of end joining. The
frequency and spectrum of repair events in each genetic background
reflects the competition between available DSB repair pathways. This
assay can provide a simple approach to identifying genetic and
environmental factors that influence chromosomal stability.
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