Frequency of DNA end joining <i>in trans</i> is not determined by the predamage spatial proximity of double-strand breaks in yeast.

Abstract

DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure <i>cis</i> (same site) vs. <i>trans</i> (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ <i>in trans</i> led to expression of a <i>LEU2</i> reporter gene. Consistent with prior reports, <i>cis</i> NHEJ was more efficient than <i>trans</i> NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, <i>trans</i> NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two <i>trans</i> repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and <i>trans</i> repair frequency, but still without a strong correlation of <i>trans</i> repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.