Abstract
DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repair-associated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Association of the single-stranded telomere binding protein Cdc13 with a SiRTA is required to stimulate de novo telomere addition. Because extensive resection must occur prior to Cdc13 binding, we utilized these sites to monitor the effect of proteins involved in homologous recombination. We find that telomere addition is significantly reduced in the absence of the Rad51 recombinase, while loss of Rad52, required for Rad51 nucleoprotein filament formation, has no effect. Deletion of RAD52 suppresses the defect of the rad51Δ strain, suggesting that Rad52 inhibits de novo telomere addition in the absence of Rad51. The ability of Rad51 to counteract this effect of Rad52 does not require DNA binding by Rad51, but does require interaction between the two proteins, while the inhibitory effect of Rad52 depends on its interaction with Replication Protein A (RPA). Intriguingly, the genetic interactions we report between RAD51 and RAD52 are similar to those previously observed in the context of checkpoint adaptation. Forced recruitment of Cdc13 fully restores telomere addition in the absence of Rad51, suggesting that Rad52, through its interaction with RPA-coated single-stranded DNA, inhibits the ability of Cdc13 to bind and stimulate telomere addition. Loss of the Rad51-Rad52 interaction also stimulates a subset of Rad52-dependent microhomology-mediated repair (MHMR) events, consistent with the known ability of Rad51 to prevent single-strand annealing.
Highlights
The ends of most linear eukaryotic chromosomes are organized into special nucleoprotein structures, telomeres, that are essential for the maintenance of genome stability and integrity
DNA double-strand breaks (DSBs) can lead to chromosome loss and rearrangement associated with cancer and genetic disease, so understanding how the cell coordinates multiple possible repair pathways is of critical importance
We show that interactions between proteins with known roles during DSB repair modulate the probability of telomerase action at hotspots of de novo telomere addition in the yeast genome by influencing the association of Cdc13, a protein required for telomerase recruitment, with sites of telomere addition
Summary
The ends of most linear eukaryotic chromosomes are organized into special nucleoprotein structures, telomeres, that are essential for the maintenance of genome stability and integrity. In association with telomere binding proteins, telomeres protect the ends of the chromosomes from being recognized as DNA double-strand breaks (DSBs), thereby preventing inappropriate nucleolytic processing, fusion, and recombination [1]. The telomerase reverse transcriptase utilizes its intrinsic RNA component as a template for the addition of TG-rich sequence repeats (TG1-3 in yeast) to the overhanging 3’ strand at the chromosome terminus [2]. Association of yeast Rad with RPA-coated single-stranded DNA results in the displacement of RPA and formation of the Rad nucleoprotein filament [3,7,8] that initiates the homology search and coordinates strand invasion into homologous duplex DNA [3,9]. In addition to serving as a mediator of Rad filament formation, Rad facilitates annealing between complementary RPA-coated single strands [10,11]
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