RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae

Abstract

Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of doublestrand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly-efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300 bp and 60 bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in <em>Saccharomyces cerevisiae</em>

“…Eukaryotic genomes are replete with repetitive sequences that are excellent substrates for generating translocations and other genomic rearrangements 20,21 . Chromosomal translocations by HR are frequently observed when DSBs are introduced between repetitive sequences 12,21,22 . Overwhelming evidence suggests that much of the genomic instability associated with leukemias and lymphomas can be attributed to chromosomal translocations, highlighting the importance of understanding how this mechanism occurs in eukaryotes 22,23 .…”

“…Careful analysis has allowed us to gather additional important information about the assay 12 . Genomic Southern blot analysis has provided evidence that there is virtually complete cutting of chromosomes XV and III after 30 minutes of HO-endonuclease induction, and thus there is no significant background of uncut chromosomal substrates in the population (G. Manthey & A. Bailis, unpublished results).…”

“…The his3-Δ5' substrate is located at the LEU2 locus on one copy of chromosome III, with the other copy of chromosome III containing an unaltered LEU2 allele (Figure 1 cleavage by inducing expression of the HO gene through the addition of galactose to the medium. After HO-endonuclease induced cleavage at the his3-Δ3' and his3-Δ5' substrates, cells can efficiently use the shared short tract of HIS3 sequence homology (311 bp or 60 bp) to repair the broken chromosomes by HR, generating a translocation chromosome with an intact HIS3 allele [12][13][14]25 .…”

“…Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium ( Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system [12][13][14] . …”

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

“…Unlike Rad52, the annealing activity of Rad59 is not stimulated by RPA (Petukhova et al 1999;Wu et al 2006). Rad59 also lacks recombination mediator activity, but it is biologically important in recombination pathways that do not require Rad51, such as SSA (Sugawara et al 2000;Pannunzio et al 2008Pannunzio et al , 2010. Rad59 conceivably acts as an accessory protein for annealing reactions promoted by Rad52 during the second-end capture phases of dHJ and SDSA.…”